U.S. patent application number 13/836168 was filed with the patent office on 2014-04-17 for macrocyclic indole derivatives useful as hepatitis c virus inhibitors.
This patent application is currently assigned to JANSSEN R&D IRELAND. The applicant listed for this patent is Janssen R&D Ireland. Invention is credited to Katie Ingrid Eduard Amssoms, Tse-I Lin, Pierre Jean-Marie Bernard Raboisson, Abdellah Tahri, Sandrine Marie Helene Vendeville.
Application Number | 20140107101 13/836168 |
Document ID | / |
Family ID | 40972916 |
Filed Date | 2014-04-17 |
United States Patent
Application |
20140107101 |
Kind Code |
A1 |
Vendeville; Sandrine Marie Helene ;
et al. |
April 17, 2014 |
Macrocyclic Indole Derivatives Useful as Hepatitis C Virus
Inhibitors
Abstract
Inhibitors of HCV replication of formula (I) ##STR00001##
including stereochemically isomeric forms, and salts, hydrates,
solvates thereof, wherein R.sup.1, R.sup.2, R.sup.4, R.sup.5,
R.sup.6 and R.sup.7 have the meaning defined in the claims. The
present invention also relates to processes for preparing said
compounds, pharmaceutical compositions containing them and their
use in HCV therapy.
Inventors: |
Vendeville; Sandrine Marie
Helene; (Mechelen, BE) ; Raboisson; Pierre Jean-Marie
Bernard; (Mechelen, BE) ; Lin; Tse-I;
(Mechelen, BE) ; Tahri; Abdellah; (Anderlecht,
BE) ; Amssoms; Katie Ingrid Eduard; (Hove,
BE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Janssen R&D Ireland; |
|
|
US |
|
|
Assignee: |
JANSSEN R&D IRELAND
Little Island
IL
|
Family ID: |
40972916 |
Appl. No.: |
13/836168 |
Filed: |
March 15, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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13000583 |
Dec 21, 2010 |
|
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PCT/EP2009/004942 |
Jul 8, 2009 |
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13836168 |
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Current U.S.
Class: |
514/214.02 ;
540/456 |
Current CPC
Class: |
C07D 513/14 20130101;
C07D 515/18 20130101; A61P 31/12 20180101; A61K 45/06 20130101;
A61P 31/18 20180101; A61P 31/14 20180101; A61P 31/20 20180101; C07D
513/22 20130101; A61P 43/00 20180101; A61K 31/55 20130101; C07D
513/18 20130101 |
Class at
Publication: |
514/214.02 ;
540/456 |
International
Class: |
C07D 515/18 20060101
C07D515/18; C07D 513/18 20060101 C07D513/18; A61K 45/06 20060101
A61K045/06; A61K 31/55 20060101 A61K031/55; C07D 513/22 20060101
C07D513/22 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 8, 2008 |
EP |
08159965.6 |
Jul 11, 2008 |
EP |
08160254.2 |
Aug 4, 2008 |
EP |
08161743.3 |
Claims
1. A method for inhibiting HCV replication, comprising
administering to a subject in need thereof a compound of Formula
(I) ##STR00212## or a stereochemically isomeric form, N-oxide,
salt, hydrate, or solvate thereof, wherein: R.sup.1 is a bivalent
chain selected from ##STR00213## each R.sup.3 is independently
selected from the group comprising hydrogen, C.sub.1-4alkyl and
C.sub.3-5cycloalkyl; a is 3, 4, 5 or 6; each b is independently 1
or 2; c is 1 or 2; macrocycle A has 14 to 18 member atoms, in
particular macrocycle A has 17 or 18 member atoms; each R.sup.2 is
independently hydrogen, halo or C.sub.1-4alkoxy; R.sup.4 and
R.sup.5 are hydrogen or R.sup.4 and R.sup.5 together form a double
bond or a methylene group to form a fused cyclopropyl; R.sup.6 is
hydrogen or methyl; and R.sup.7 is a C.sub.3-7cycloalkyl optionally
substituted with halo.
2. The method of claim 1, wherein the wherein, R.sup.1 is selected
from --N(R.sup.3)--(CH.sub.2).sub.4--N(R.sup.3)--, ##STR00214##
and, each R.sup.3 is independently selected from hydrogen and
methyl.
3. The method of claim 1, wherein R.sup.2 is positioned the benzene
group in para with respect to the bond linking this benzene to the
indole group.
4. The method of claim 1 wherein R.sup.2 is selected from fluoro
and methoxy.
5. The method of claim 1 wherein R.sup.7 is selected from
cyclohexyl and 2-fluorocyclohexyl.
6. The method of claim 1 wherein R.sup.4 and R.sup.5 together form
a double bond.
7. The method of claim 1 wherein the compounds of formula (I) have
the stereochemical configuration as illustrated by formula (IA).
##STR00215##
8. The method of claim 1, wherein said compound has one of the
structural formula II-1, II-2, II-3, III-1, III-2, III-3, III-4,
IV-1, IV-2 or IV-3 ##STR00216## ##STR00217## ##STR00218##
9-15. (canceled)
16. The method of claim 1, wherein macrocycle A has 17 or 18 member
atoms.
17. The method of claim 1, wherein the compound is ##STR00219## or
a salt thereof.
18. The method of claim 17, wherein the compound is
##STR00220##
19. The method of claim 1, comprising administering to said subject
at least one other anti-HCV compound.
20. The method of claim 17, comprising administering to said
subject at least one other anti-HCV compound.
Description
FIELD OF THE INVENTION
[0001] The present invention is concerned with macrocyclic indole
derivatives having inhibitory activity on the replication of the
hepatitis C virus (HCV). It further concerns compositions
comprising these compounds as active ingredients as well as
processes for preparing these compounds and compositions.
BACKGROUND OF THE INVENTION
[0002] Hepatitis C virus is the leading cause of chronic liver
disease worldwide and has become a focus of considerable medical
research. HCV is a member of the Flaviviridae family of viruses in
the hepacivirus genus, and is closely related to the flavivirus
genus, which includes a number of viruses implicated in human
disease, such as dengue virus and yellow fever virus, and to the
animal pestivirus family, which includes bovine viral diarrhoea
virus (BVDV). HCV is a positive-sense, single-stranded RNA virus,
with a genome of around 9,600 bases. The genome comprises both 5'
and 3' untranslated regions that adopt RNA secondary structures,
and a central open reading frame that encodes a single polyprotein
of around 3,010-3,030 amino acids. The polyprotein encodes ten gene
products, which are generated from the precursor polyprotein by an
orchestrated series of co- and posttranslational endoproteolytic
cleavages mediated by both host and viral proteases. The viral
structural proteins include the core nucleocapsid protein, and two
envelope glycoproteins E1 and E2. The non-structural (NS) proteins
encode some essential viral enzymatic functions (helicase,
polymerase, protease), as well as proteins of unknown function.
Replication of the viral genome is mediated by an RNA-dependent RNA
polymerase, encoded by non-structural protein 5b (NS5B). In
addition to the polymerase, the viral helicase and protease
functions, both encoded in the bifunctional NS3 protein, have been
shown to be essential for replication of HCV RNA. In addition to
the NS3 serine protease, HCV also encodes a metalloproteinase in
the NS2 region.
[0003] HCV replicates preferentially in hepatocytes but is not
directly cytopathic, leading to persistent infection. In
particular, the lack of a vigorous T-lymphocyte response and the
high propensity of the virus to mutate appear to promote a high
rate of chronic infection. There are 6 major HCV genotypes and more
than 50 subtypes, which are differently distributed geographically.
HCV type 1 is the predominant genotype in the US and Europe. For
instance, HCV type 1 accounts for 70 to 75 percent of all HCV
infections in the United States. The extensive genetic
heterogeneity of HCV has important diagnostic and clinical
implications, perhaps explaining difficulties in vaccine
development and the lack of response to therapy. An estimated 170
million persons worldwide are infected with hepatitis C virus
(HCV). Following the initial acute infection, a majority of
infected individuals develops chronic hepatitis, which can progress
to liver fibrosis leading to cirrhosis, end-stage liver disease,
and HCC (hepatocellular carcinoma) (National Institutes of Health
Consensus Development Conference Statement: Management of Hepatitis
C. Hepatology, 36, 5 Suppl. S3-S20, 2002). Liver cirrhosis due to
HCV infection is responsible for about 10,000 deaths per year in
the U.S.A. alone, and is the leading cause for liver
transplantations. Transmission of HCV can occur through contact
with contaminated blood or blood products, for example following
blood transfusion or intravenous drug use. The introduction of
diagnostic tests used in blood screening has led to a downward
trend in post-transfusion HCV incidence. However, given the slow
progression to the end-stage liver disease, the existing infections
will continue to present a serious medical and economic burden for
decades (Kim, W. R. Hepatology, 36, 5 Suppl. S30-S34, 2002).
[0004] Current HCV therapies are based on (pegylated)
interferon-alpha (IFN-.alpha.) in combination with ribavirin. This
combination therapy yields a sustained virologic response in more
than 40% of patients infected by genotype 1 viruses and about 80%
of those infected by genotypes 2 and 3. Beside the limited efficacy
on HCV type 1, combination therapy has significant side effects and
is poorly tolerated in many patients. For instance, in registration
trials of pegylated interferon and ribavirin, significant side
effects resulted in discontinuation of treatment in approximately
10 to 14 percent of patients. Major side effects of combination
therapy include influenza-like symptoms, hematologic abnormalities,
and neuropsychiatric symptoms. The development of more effective,
convenient and tolerated treatments is a major public health
objective. Thus, the treatment of this chronic disease is an unmet
clinical need, since current therapy is only partially effective
and limited by undesirable side effects.
[0005] One area of particular focus has been the search for
inhibitors of the NS5b RNA-dependent RNA polymerase (RdRp). Close
structural homologs of this polymerase do not exist within the
uninfected host cell and the finding of inhibitors of said
polymerase would provide a more specific mode of action. Inhibitors
that are currently under investigation can be classified as either
nucleoside inhibitors (NIs) or non-nucleoside inhibitors (NNIs).
NIs directly compete with nucleotide substrates for binding to
highly conserved active sites. Greater specificity may be achieved
by NNIs, which may interact outside of the highly conserved active
site at a unique allosteric site common only to structurally
related polymerases.
[0006] Indole derivatives have been described for HCV inhibitory
activity. WO 2007/092000 discloses tetracyclic indole derivatives
as HCV NS5B inhibitors for the treatment and/or prevention of HCV
virus infection. US 2008/0146537 discloses cyclopropyl fused
indolobenzazepine HCV NS5B inhibitors. WO 2008/075103 discloses
macrocyclic indole derivatives useful for the treatment or
prevention of infection by hepatitis C virus.
[0007] To date, preliminary clinical trials have resulted in a high
failure rate, thereby highlighting the need to pursue the search
for novel NS5b inhibitors. There is a high medical need for safe
and effective anti-HCV treatment. Such HCV inhibitors may overcome
the disadvantages of current HCV therapy such as side effects,
limited efficacy, the emergence of resistance, and compliance
failures, as well as improve the sustained viral response. In
particular wherein the therapeutic compounds have good
bioavailability and a favorable pharmacokinetic and metabolic
profile.
SUMMARY OF THE INVENTION
[0008] It has been found that certain macrocyclic indole
derivatives exhibit antiviral activity in subjects infected with
HCV with useful properties regarding one or more of the following
parameters: antiviral efficacy, favorable mutant prophile, lack of
toxicity, favorable pharmacokinetic and metabolic profile, and ease
of formulation and administration. These compounds are therefore
useful in treating or combating HCV infections.
[0009] The present invention concerns inhibitors of HCV
replication, which can be represented by formula (I),
##STR00002##
including stereochemically isomeric forms, and N-oxides, salts,
hydrates, and solvates thereof, wherein: [0010] R.sup.1 is a
bivalent chain selected from
[0010] ##STR00003## [0011] each R.sup.3 is independently selected
from the group comprising hydrogen, C.sub.1-4alkyl and
C.sub.3-5cycloalkyl; [0012] a is 3, 4, 5 or 6; [0013] each b is
independently 1 or 2; [0014] c is 1 or 2; [0015] macrocycle A has
14 to 18 member atoms; [0016] each R.sup.2 is independently
hydrogen, halo or C.sub.1-4alkoxy; [0017] R.sup.4 and R.sup.5 are
hydrogen or R.sup.4 and R.sup.5 together form a double bond or a
methylene group to form a fused cyclopropyl; [0018] R.sup.6 is
hydrogen or methyl; and [0019] R.sup.7 is a C.sub.3-7cycloalkyl
optionally substituted with halo.
[0020] The invention further relates to methods for the preparation
of the compounds of formula (I), including stereochemically
isomeric forms, and N-oxides, quaternary amines, metal complexes,
salts, hydrates or solvates thereof, their intermediates, and the
use of the intermediates in the preparation of the compounds of
formula (I). The invention relates to the compounds of formula (I)
per se, including stereochemically isomeric forms, and N-oxides,
quaternary amines, metal complexes, salts, hydrates or solvates
thereof, for use as a medicament. The invention relates to the
compounds of formula (I) per se, including stereochemically
isomeric forms, and N-oxides, quaternary amines, metal complexes,
salts, hydrates or solvates thereof, for treating hepatitis C. The
invention further relates to pharmaceutical compositions comprising
a carrier and an anti-virally effective amount of a compound of
formula (I) as specified herein. The pharmaceutical compositions
may comprise combinations of the aforementioned compounds with
other anti-HCV agents. The pharmaceutical compositions may comprise
combinations of the aforementioned compounds with anti-HIV agents.
The invention further relates to the aforementioned pharmaceutical
compositions for administration to a subject suffering from HCV
infection.
[0021] The invention also relates to the use of a compound of
formula (I), including stereochemically isomeric forms, or
N-oxides, quaternary amines, metal complexes, salts, hydrates or
solvates thereof, for the manufacture of a medicament for
inhibiting HCV replication. The invention also relates to the use
of a compound of formula (I), including stereochemically isomeric
forms, or N-oxides, quaternary amines, metal complexes, salts,
hydrates or solvates thereof, for the manufacture of a medicament
for preventing or treating conditions associated with HCV. The
invention also relates to a method of inhibiting HCV replication in
a warm-blooded animal said method comprising the administration of
an effective amount of a compound of formula (I), including
stereochemically isomeric forms, or N-oxides, quaternary amines,
metal complexes, salts, hydrates or solvates thereof. The invention
also relates to a method for preventing or treating conditions
associated with HCV in a warm-blooded animal said method comprising
the administration of an effective amount of a compound of formula
(I), including stereochemically isomeric forms, or N-oxides,
quaternary amines, metal complexes, salts, hydrates or solvates
thereof.
DETAILED DESCRIPTION
[0022] The present invention will now be further described. In the
following passages, different aspects or embodiments of the
invention are defined in more detail. Each aspect or embodiment so
defined may be combined with any other aspect(s) or embodiment(s)
unless clearly indicated to the contrary. In particular, any
feature indicated as being preferred or advantageous may be
combined with any other feature or features indicated as being
preferred or advantageous to formulate a particular embodiment.
[0023] As used in the foregoing and hereinafter, the following
definitions apply unless otherwise noted.
[0024] For the purpose of the present invention, the terms
"subject" or "infected subject" or "patient" refers to an
individual infected with HCV, in need of treatment.
[0025] The term "halo" or "halogen" is generic to fluoro, chloro,
bromo and iodo.
[0026] As used herein "C.sub.1-4alkyl" as a group or part of a
group defines straight or branched chain saturated hydrocarbon
radicals having from 1 to 4 carbon atoms such as for example
methyl, ethyl, prop-1-yl, prop-2-yl, but-1-yl, but-2-yl, isobutyl,
2-methylprop-1-yl; "C.sub.1-3alkyl" as a group or part of a group
defines straight or branched chain saturated hydrocarbon radicals
having from 1 to 3 carbon atoms such as for example methyl, ethyl,
prop-1-yl, prop-2-yl.
[0027] The term "C.sub.1-6alkylene" as a group or part of a group
refers to C.sub.1-6alkyl groups that are divalent, i.e., with two
single bonds for attachment to two other groups. Non-limiting
examples of alkylene groups includes methylene, ethylene,
methylmethylene, propylene, ethylethylene, 1-methylethylene and
1,2-dimethylethylene. "C.sub.3-7cycloalkyl" is generic to
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.
The term "C.sub.3-5cycloalkyl" is meant to comprise cyclopropyl,
cyclobutyl and cyclopentyl.
[0028] The term "C.sub.1-4alkoxy" or "C.sub.1-4alkyloxy" as a group
or part of a group refers to a radical having the Formula
--OR.sup.a wherein R.sup.a is C.sub.1-4alkyl as defined above.
Non-limiting examples of suitable C.sub.1-4alkoxy include methoxy,
ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy and
tert-butoxy.
[0029] It should be noted that the radical positions on any
molecular moiety used in the definitions may be anywhere on such
moiety as long as it is chemically stable.
[0030] Radicals used in the definitions of the variables include
all possible isomers unless otherwise indicated. For instance,
piperidinyl includes piperidin-1-yl, piperidin-2-yl,
piperidin-3-yl, and piperidin-4-yl; pentyl includes pent-1-yl,
pent-2-yl and pent-3-yl.
[0031] When any variable occurs more than one time in any
constituent, each definition is independent.
[0032] Whenever used hereinafter, the term "compounds of formula
(I)", or "the present compounds" or similar terms, it is meant to
include the compounds of formula (I), including stereochemically
isomeric forms, and their N-oxides, quaternary amines, metal
complexes, salts, hydrates or solvates thereof. One embodiment
comprises the compounds of formula (I) or any subgroup thereof
specified herein, including the possible stereochemically isomeric
forms, as well as the N-oxides, salts, hydrates, and solvates
thereof. Another embodiment comprises the compounds of formula (I)
or any subgroups thereof specified herein, including the possible
stereochemically isomeric forms, as well as the N-oxides, salts,
hydrates, and solvates thereof.
[0033] Whenever used hereinafter, the term "optionally substituted"
is meant to include unsubstituted as well as substituted with at
least one of the specified substituting radicals. For the purpose
of example, "C.sub.1-4alkyl optionally substituted with chloro" is
meant to include unsubstituted C.sub.1-4alkyl as well as
C.sub.1-4alkyl substituted with chloro.
[0034] The compounds of formula (I) may have one or more centers of
chirality and may exist as stereochemically isomeric forms. The
term "stereochemically isomeric forms" as used herein defines all
the possible compounds made up of the same atoms bonded by the same
sequence of bonds but having different three-dimensional
structures, which the compounds of formula (I) may possess.
[0035] With reference to the instances where (R) or (5) is used to
designate the absolute configuration of a chiral atom within a
substituent, the designation is done taking into consideration the
whole compound and not the substituent in isolation.
[0036] In one aspect, the present invention provides compounds of
formula (I)
##STR00004##
including stereochemically isomeric forms, and N-oxides, salts,
hydrates, and solvates thereof, wherein R.sup.1, R.sup.2, R.sup.4,
R.sup.5, R.sup.6, R.sup.7 and A have the same meaning as defined
herein. Embodiments of the present inventions concerns compounds of
formula (I), or any subgroup thereof as defined herein, wherein one
or more of the definitions for R.sup.1, R.sup.2, R.sup.4, R.sup.5,
R.sup.6, and R.sup.7 as specified in the embodiments herein-under
apply:
[0037] Particular subgroups of the compounds of formula (I) are
compounds of formula (II), (III) or (IV)
##STR00005##
wherein R.sup.1, R.sup.2, R.sup.4, R.sup.5, R.sup.6, R.sup.7 and A
have the same meaning as defined herein.
[0038] In one embodiment, R.sup.1 is a bivalent chain selected
from
##STR00006##
In a particular embodiment, R.sup.1 is selected from
##STR00007##
wherein a and c are as defined herein above, or wherein a is 4 or 5
and c is 1 or 2. In another particular embodiment R.sup.1 is
selected from --N(R.sup.3)--(CH.sub.2).sub.4--N(R.sup.3)--,
##STR00008##
When R.sup.1 is
##STR00009##
[0039] it is understood R.sup.1 may be oriented in two directions,
i.e. the piperazinyl moiety may be connected to the sulfonamide
group while the aliphatic amine is connected to the carbonyl group,
or, the piperazinyl moiety is connected to the carbonyl group and
the aliphatic amine is connected to the sulfonamide group.
Preferably, when R.sup.1 is
##STR00010##
the piperazinyl moiety is connected to the carbonyl group and the
aliphatic amine is connected to the sulfonamide group.
[0040] In a more particular embodiment, R.sup.1 is selected
from
##STR00011##
[0041] Alternatively, R.sup.1 is selected from
##STR00012##
[0042] In a preferred embodiment, R.sup.1 is
##STR00013##
[0043] In another embodiment, R.sup.1 is
##STR00014##
[0044] In another embodiment R.sup.1 is
##STR00015##
[0045] In another preferred embodiment, R.sup.1 is
##STR00016##
[0046] Each R.sup.3 is independently selected from the group
comprising hydrogen, C.sub.1-4alkyl and C.sub.3-5cycloalkyl. In a
particular embodiment, R.sup.3 is independently selected from the
group consisting of hydrogen, methyl, ethyl, isopropyl and
cyclopropyl. In a more particular embodiment, each R.sup.3 is
independently selected from the group consisting of hydrogen and
methyl; or, R.sup.3 is methyl.
[0047] Macrocycle A has 14 to 18 member atoms. In a particular
embodiment, macrocycle A has 16, 17 or 18 member atoms. In a more
particular embodiment, A has 17 member atoms.
[0048] R.sup.2 is selected from the group comprising hydrogen, halo
or C.sub.1-4alkoxy. In a particular embodiment, R.sup.2 is selected
from the group comprising hydrogen, chloro, fluoro or methoxy. In a
more particular embodiment, R.sup.2 is hydrogen or methoxy or
chloro; or, alternatively, R.sup.2 is fluoro or methoxy; or, in a
preferred embodiment, R.sup.2 is methoxy. In another embodiment,
R.sup.2 is positioned on the benzene ring in meta or para with
respect to the bond linking the benzene to the indole group. In a
preferred embodiment, R.sup.2 is positioned on the benzene ring in
para with respect to the bond linking this benzene to the indole
group.
[0049] R.sup.4 and R.sup.5 are hydrogen or R.sup.4 and R.sup.5
together form a double bond or a methylene group to form a fused
cyclopropyl. In a particular embodiment, R.sup.4 and R.sup.5 are
hydrogen or R.sup.4 and R.sup.5 together form a methylene group to
form a fused cyclopropyl.
[0050] In another particular embodiment, R.sup.4 and R.sup.5
together form a double bond.
[0051] In another embodiment, R.sup.6 is selected from hydrogen and
methyl. In a particular embodiment, R.sup.6 is hydrogen when the
compound of formula (I) is a compound of formula (III) or (IV). In
another particular embodiment, R.sup.6 is methyl when the compound
of formula (I) is a compound of formula (II).
[0052] R.sup.7 is a C.sub.3-7cycloalkyl optionally substituted with
halo. In a particular embodiment, R.sup.7 is selected from
cyclopentyl, cyclohexyl, and fluorocyclohexyl (in particular,
2-fluorocyclohexyl). In a preferred embodiment, R.sup.7 is
cyclohexyl.
[0053] A particular subgroup of compounds of formula (I) are
compounds of formula (I) wherein R.sup.4 and R.sup.5 together form
a double bond, and wherein one or more of the definitions for
R.sup.1, R.sup.2, R.sup.6, and R.sup.7 as specified in the
embodiments herein apply. A more particular subgroup of compounds
of formula (I) are compounds of formula (II), wherein R.sup.1,
R.sup.2, R.sup.4, R.sup.5, R.sup.6, R.sup.7 and A have the same
meaning as defined herein. More particular are those compound
represented by the following structural formulae (II-1), (II-2),
and (II-3) wherein R.sup.2, R.sup.6 and R.sup.7 have the same
meaning as defined herein for compounds of formula (I) or subgroups
thereof
##STR00017##
[0054] In a particular embodiment, the invention provides compounds
of, independently, formula (II), (II-1), (II-2) and (II-3) wherein
R.sup.6 is hydrogen or methyl, more in particular, wherein R.sup.6
is a methyl.
[0055] In another embodiment, the invention provides compounds of
formula (II) or subgroups thereof wherein R.sup.7 is cyclohexyl or
2-fluorocyclohexyl.
[0056] In another embodiment, the invention provides compounds of
formula (II) or subgroups thereof wherein R.sup.2 is hydrogen,
methoxy or chloro. Alternatively, the invention provides compounds
of formula (II) or subgroups thereof wherein R.sup.2 is fluoro or
methoxy.
[0057] A particular subgroup of compounds of formula (I) are
compounds of formula (III), wherein R.sup.1, R.sup.2, R.sup.4,
R.sup.5, R.sup.6, R.sup.7 and A have the same meaning as defined
herein. More particular are those compounds represented by the
following structural formulae (III-1), (III-2), (III-3) and (III-4)
wherein R.sup.2, R.sup.6 and R.sup.7 have the same meaning as
defined herein for compounds of formula (I).
##STR00018##
[0058] In particular, the invention provides compounds of,
independently, formula (III), (III-1), (III-2), (III-3) and (III-4)
wherein R.sup.6 is hydrogen.
[0059] In another embodiment, the invention provides compounds of
formula (III) or subgroups thereof wherein R.sup.7 is cyclohexyl or
2-fluorocyclohexyl.
[0060] In another embodiment, the invention provides compounds of
formula (III) or subgroups thereof wherein R.sup.2 is hydrogen,
methoxy or chloro.
[0061] A particular subgroup of compounds of formula (I) are
compounds of formula (IV), wherein R.sup.1, R.sup.2, R.sup.4,
R.sup.5, R.sup.6, R.sup.7 and A have the same meaning as defined
herein. More particular are those compound represented by the
following structural formulae (IV-1), (IV-2), and (IV-3) wherein
R.sup.2, R.sup.6 and R.sup.7 have the same meaning as defined
herein for compounds of formula (I).
##STR00019##
[0062] In another embodiment, the invention provides compounds of
formula (IV) or subgroups thereof wherein R.sup.7 is cyclohexyl or
2-fluorocyclohexyl.
[0063] In another embodiment, the invention provides compounds of
formula (IV) or subgroups thereof wherein R.sup.2 is hydrogen,
methoxy or chloro.
[0064] In a particular embodiment, the present invention concerns
compounds of formula (II-1), (III-1), and (IV-1). Another
embodiment of the present invention concerns compounds of formula
(II-2), (III-2) and (IV-2). Another embodiment of the present
invention concerns compounds of formula (II-3), (III-3) and
(IV-3).
[0065] In a particular embodiment, the invention provides compounds
of formula (I) selected from the group comprising
##STR00020## ##STR00021## ##STR00022## ##STR00023##
##STR00024##
[0066] More in particular, the present invention provides compounds
of formula (I) selected from
##STR00025## ##STR00026## ##STR00027##
[0067] Alternatively, the present invention provides compounds of
formula (I) selected from
##STR00028## ##STR00029## ##STR00030## ##STR00031## ##STR00032##
##STR00033##
[0068] More in particular, the present invention provides compounds
of formula (I) selected from
##STR00034##
[0069] Unless otherwise mentioned or indicated, the chemical
designation of a compound encompasses the mixture of some or all
possible stereochemically isomeric forms, which said compound might
possess. Said mixture may contain all diastereomers and/or
enantiomers of the basic molecular structure of said compound. All
stereochemically isomeric forms of the compounds of the present
invention both in pure form or mixed with each other are intended
to be embraced within the scope of the present invention.
[0070] Pure stereoisomeric forms of the compounds and intermediates
as mentioned herein are defined as isomers substantially free of
other enantiomeric or diastereomeric forms of the same basic
molecular structure of said compounds or intermediates. In
particular, the term "stereoisomerically pure" concerns compounds
or intermediates having a stereoisomeric excess of at least 80%
(i.e. minimum 90% of one isomer and maximum 10% of the other
possible isomers) up to a stereoisomeric excess of 100% (i.e. 100%
of one isomer and none of the other), more in particular, compounds
or intermediates having a stereoisomeric excess of 90% up to 100%,
even more in particular having a stereoisomeric excess of 94% up to
100% and most in particular having a stereoisomeric excess of 97%
up to 100%. The terms "enantiomerically pure" and
"diastereomerically pure" should be understood in a similar way,
but then having regard to the enantiomeric excess, and the
diastereomeric excess, respectively, of the mixture in
question.
[0071] Pure stereoisomeric forms of the compounds and intermediates
of this invention may be obtained by the application of art-known
procedures. For instance, enantiomers may be separated from each
other by the selective crystallization of their diastereomeric
salts with optically active acids or bases. Examples thereof are
tartaric acid, dibenzoyltartaric acid, ditoluoyltartaric acid and
camphosulfonic acid. Alternatively, enantiomers may be separated by
chromatographic techniques using chiral stationary phases. Said
pure stereochemically isomeric forms may also be derived from the
corresponding pure stereochemically isomeric forms of the
appropriate starting materials, provided the reaction occurs
stereospecifically. Preferably, if a specific stereoisomer is
desired, said compound is synthesized by stereospecific methods of
preparation. These methods will advantageously employ
enantiomerically pure starting materials.
[0072] The diastereomeric racemates of the compounds of formula (I)
or any subgroup thereof, can be obtained separately by conventional
methods. Appropriate physical separation methods that may
advantageously be employed are, for example, selective
crystallization and chromatography, e.g. column chromatography.
[0073] For some of the compounds of formula (I), their N-oxides,
salts, hydrates, solvates, quaternary amines, or metal complexes,
and the intermediates used in the preparation thereof, the absolute
stereochemical configuration was not experimentally determined. A
person skilled in the art is able to determine the absolute
configuration of such compounds using art-known methods such as,
for example, X-ray diffraction.
[0074] In an embodiment, the present invention concerns compounds
of formula (IIIA), (IIIB), (IVA) and (IVB),
##STR00035##
wherein R.sup.1, R.sup.2, R.sup.6, R.sup.7 and A have the same
meaning as that defined herein.
[0075] In a more particular embodiment, the present invention
concerns compounds of Formula (IIIA-1), (IIIA-2), (IIIA-3),
(IIIA-4), (IIIB-1), (IIIB-2), (IIIB-3), (IIIB-4), (IVA-1), (IVA-2),
(IVA-3), (IVB-1), (IVB-2) and (IVB-3)
##STR00036## ##STR00037## ##STR00038## ##STR00039##
wherein R.sup.2, R.sup.6 and R.sup.7 have the same meaning as that
defined herein.
[0076] In another embodiment, where applicable, compounds of
formula (I) or subgroups thereof have the stereochemical
configuration as illustrated by formula (IA).
##STR00040##
[0077] The present invention is also intended to include all
isotopes of atoms occurring on the present compounds. Isotopes
include those atoms having the same atomic number but different
mass numbers. By way of general example and without limitation,
isotopes of hydrogen include tritium and deuterium. Isotopes of
carbon include C-13 and C-14.
[0078] For therapeutic use, salts of the compounds of formula (I)
are those wherein the counter-ion is pharmaceutically acceptable.
However, salts of acids and bases, which are non-pharmaceutically
acceptable, may also find use, for example, in the preparation or
purification of a pharmaceutically acceptable compound. All salts,
whether pharmaceutically acceptable or not are included within the
ambit of the present invention.
[0079] The pharmaceutically acceptable acid and base salts as
mentioned hereinabove are meant to comprise the therapeutically
active non-toxic acid and base addition salt forms that the
compounds of formula (I) are able to form. The pharmaceutically
acceptable acid addition salts can conveniently be obtained by
treating the base form with such appropriate acid. Appropriate
acids comprise, for example, inorganic acids such as hydrohalic
acids, e.g. hydrochloric or hydrobromic acid, sulfuric, nitric,
phosphoric and the like acids; or organic acids such as, for
example, acetic, propanoic, hydroxyacetic, lactic, pyruvic, oxalic
(i.e. ethanedioic), malonic, succinic (i.e. butanedioic acid),
maleic, fumaric, malic (i.e. hydroxybutanedioic acid), tartaric,
citric, methanesulfonic, ethanesulfonic, benzenesulfonic,
p-toluenesulfonic, cyclamic, salicylic, p-amino-salicylic, pamoic
and the like acids.
[0080] Conversely, said salt forms can be converted by treatment
with an appropriate base into the free base form.
[0081] The compounds of formula (I) or any subgroup thereof
containing an acidic proton may also be converted into their
non-toxic metal or amine addition salt forms by treatment with
appropriate organic and inorganic bases. Appropriate base salt
forms comprise, for example, the ammonium salts, the alkali and
earth alkaline metal salts, e.g. the lithium, sodium, potassium,
magnesium, calcium salts and the like, salts with organic bases,
e.g. the benzathine, N-methyl-D-glucamine, hydrabamine salts, and
salts with amino acids such as, for example, arginine, lysine and
the like.
[0082] The term "quaternary amine" as used hereinbefore defines the
quaternary ammonium salts which the compounds of formula (I) or any
subgroup thereof are able to form by reaction between a basic
nitrogen of a compound of formula (I) or any subgroup thereof and
an appropriate quaternizing agent, such as, for example, an
optionally substituted alkylhalide, arylhalide or arylalkylhalide,
e.g. methyliodide or benzyliodide. Other reactants with good
leaving groups may also be used, such as alkyl
trifluoromethanesulfonates, alkyl methanesulfonates, and alkyl
p-toluenesulfonates. A quaternary amine has a positively charged
nitrogen. Pharmaceutically acceptable counterions include chloro,
bromo, iodo, trifluoroacetate and acetate. The counterion of choice
can be introduced using ion exchange resins.
[0083] The N-oxide forms of the present compounds are meant to
comprise the compounds of formula (I) or any subgroup thereof
wherein one or several nitrogen atoms are oxidized to the so-called
N-oxide.
[0084] It will be appreciated that the compounds of formula (I) or
any subgroup thereof may have metal binding, chelating, complex
forming properties and therefore may exist as metal complexes or
metal chelates. Such metalated derivatives of the compounds of
formula (I) or any subgroup thereof are intended to be included
within the scope of the present invention.
[0085] Some of the compounds of formula (I) or any subgroup thereof
and intermediates may also exist in one or more tautomeric form.
Such forms although not explicitly indicated in the above formula
are intended to be included within the scope of the present
invention. Accordingly, the compounds and intermediates may be
present as a mixture of tautomers or as an individual tautomer.
[0086] In the invention, particular preference is given to
compounds of Formula I or any subgroup thereof, that in the
inhibition assays described below have an inhibition value of less
than 100 .mu.M, preferably less than 50 .mu.M, more preferably less
than 10 .mu.M, preferably less than 5 .mu.M, even more preferably
less than 1 .mu.M preferably less than 100 nM, and in particular
less than 10 nM, as determined by a suitable assay, such as the
assays used in the Examples below.
[0087] It is to be understood that the above defined subgroups of
compounds of formula (I) as well as any other subgroup defined
herein, are meant to include stereochemically isomeric forms, and
any N-oxides, salts, quaternary amines, hydrates, solvates and
metal complexes of such compounds.
Preparation of the Compounds of Formula (I)
General Synthetic Schemes
[0088] Compounds of formula (I) may be prepared following the
different methods A, B, C, D, E, F and G described below, from
indole derivatives A-1
##STR00041##
wherein R.sup.2, R.sup.4, R.sup.5, R.sup.6 and R.sup.7 are as
defined for compounds of formula (I) or subgroups thereof, and Ra
is selected from methyl and tert-butyl and Rb is selected from
methyl. The compounds of formula (A-1) are either known in the art
or may be obtained as described in US20070270406A1, WO2007/054741
and WO2007/092000.
Method A
[0089] A schematic overview for the synthesis of the compounds of
formula (I) is given in scheme 1. The method starts from a compound
of formula A-1.
[0090] Compounds of formula A-2 may be prepared by the
regioselective hydrolysis of the ester bearing the Rb group, under
basic conditions, using a hydroxide such as LiOH or NaOH, in polar
solvents such as water, an alcohol such as methanol or ethanol,
tetrahydrofurane (THF), or a mixture thereof. This method may be
used when Rb is a methyl group and Ra is a tert-butyl group, or Ra
is a methyl group.
[0091] A monoprotected bifunctional R.sup.1 derived reagent of
formula PG-R.sup.1--H wherein R.sup.1 is as defined for formula (I)
or subgroups thereof, may then be coupled to the carboxylic acid of
compounds A-2 to form an amide bond, leading to compounds A-3.
"PG", as used herein, is a suitable amine protecting group, chosen
from the ones known in the art. Preferably PG is a
tert-butyloxycarbonyl (Boc) protecting group or a
4-nitrobenzenesulfonyl (nosyl) group.
[0092] The formation of amide bonds can be carried out using
standard procedures such as those used for coupling amino acids in
peptide synthesis. The latter involves the dehydrative coupling of
a carboxyl group of one reactant with an amino group of the other
reactant to form a linking amide bond. The amide bond formation may
be performed by reacting the starting materials in the presence of
a coupling agent or by converting the carboxyl functionality into
an active form such as an active ester, mixed anhydride or a
carboxyl acid chloride or bromide. General descriptions of such
coupling reactions and the reagents used therein can be found in
general textbooks on peptide chemistry, for example, M. Bodanszky,
"Peptide Chemistry", 2nd rev. ed., Springer-Verlag, Berlin,
Germany, (1993).
##STR00042## ##STR00043##
[0093] Examples of coupling reactions with amide bond formation
include the azide method, mixed carbonic-carboxylic acid anhydride
(isobutyl chloroformate) method, the carbodiimide
(dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), or
water-soluble carbodiimide such as
N-ethyl-N'[3-(dimethylamino)propyl]carbodiimide (EDC)) method, the
active ester method (e.g. p-nitrophenyl, p-chlorophenyl,
trichlorophenyl, pentachlorophenyl, pentafluorophenyl,
N-hydroxysuccinic imido and the like esters), the Woodward reagent
K-method, the 1,1-carbonyldiimidazole (CDI or
N,N'-carbonyldiimidazole) method, the phosphorus reagents or
oxidation-reduction methods. Some of these methods can be enhanced
by adding suitable catalysts, e.g. in the carbodiimide method by
adding 1-hydroxybenzotriazole, or 4-dimethylaminopyridine (4-DMAP).
Further coupling agents are
(benzotriazol-1-yloxy)-tris-(dimethylamino) phosphonium
hexafluorophosphate, either by itself or in the presence of
1-hydroxy-benzotriazole or 4-DMAP; or
2-(1H-benzotriazol-1-yl)-N,N,N',N'-tetra-methyluronium
tetrafluoroborate, or
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate. These coupling reactions can be performed in
either solution (liquid phase) or solid phase.
[0094] The coupling reactions preferably are conducted in an inert
solvent, such as halogenated hydrocarbons, e.g. dichloromethane
(DCM), chloroform, dipolar aprotic solvents such as acetonitrile,
dimethylformamide (DMF), dimethylacetamide, DMSO, HMPT, ethers such
as tetrahydrofuran (THF).
[0095] In many instances the coupling reactions are done in the
presence of a suitable base such as a tertiary amine, e.g.
triethylamine, diisopropylethylamine (DIPEA), N-methyl-morpholine,
N-methylpyrrolidine, 4-DMAP or 1,8-diazabicyclo[5.4.0]undec-7-ene
(DBU). The reaction temperature may range between 0.degree. C. and
50.degree. C. and the reaction time may range between 15 min and 24
h.
[0096] Removal of the protecting group following methods known in
the art may lead to compounds A-4. These methods include the
reaction of compounds A-3 with trifluoro acetic acid (TFA) in a
suitable solvent such as DCM, when PG is a Boc-protecting group, or
the reaction of compounds A-3 with a thiol like mercapto acetic
acid or thiophenol, in solution or in solid phase, in the presence
of a base, such as cesium carbonate or LiOH, in a suitable solvent,
such as DMF, THF when PG is nosyl. When Ra is a tert-butyl group
and PG is a Boc-protecting group, removal of PG as described above,
may lead to a compound A-4, with Ra being OH.
[0097] Compounds A-4 are then reacted with sulfamide, in a suitable
solvent, for example dioxane, under heating conditions, ie
100.degree. C. This reaction may take place under microwave
irradiation and lead to compounds A-5. Another method to introduce
the sulfamide moiety may consist of the reaction of compound A-4
with aminosulfonyl-chloride, in the presence of a suitable base,
such as triethylamine, DIPEA, or pyridine, in a suitable solvent,
such as a chlorinated solvent like DCM, or DMF, THF.
[0098] The ester function of compounds A-5, i.e. --CO--O--Ra, may
then be hydrolyzed, using conditions known in the art, and
including the saponification in basic media as described above,
leading to compounds A-6. Heating may be required to complete this
reaction. Acidic conditions may also be used to hydrolyze the ester
function of compounds A-5, for example TFA in a suitable solvent
like DCM, when Ra is a tert-butyl group.
[0099] Compounds (I) may be obtained by macrocyclisation by forming
the intramolecular acylsulfamide bond, in the presence of coupling
agents, such as CDI which converts the carboxylic acid group to a
reactive species acylimidazole, under heating. This acylimidazole
may then be purified before adding a suitable base such as DBU, in
order to perform the ring closure, which may take place under
heating conditions. Solvents used for these reactions may include
acetonitrile or THF. Other coupling agents, such as those known in
the art, may also be used to achieve the ring closure.
Method B
##STR00044##
[0101] An alternate method leading to compounds A-4 as illustrated
in scheme 2, may be the formation of an amide bond between
compounds A-2 and a symmetrical bivalent chain R1, used in excess
compared to compounds A-2. This amide bond may be synthesized as
described above, in particular using a coupling agent such as
[dimethylamino-([1,2,3]triazolo[4,5-b]pyridin-3-yloxy)-methylene]-dimethy-
l-ammonium hexafluorophosphate (HATU), in the presence of a base
such as DIPEA and in a suitable solvent like DCM, DMF, or more
preferably THF. Compounds A-4 may then be reacted as described
above in method A in order to prepare compounds (I).
Method C
##STR00045##
[0103] Compounds may be prepared directly from compounds A-2, in a
similar way as described above for the synthesis of compounds A-3,
but using a bivalent chain R.sup.1 bearing one sulfamide moiety
instead of a protecting group. Such a sulfamide chain R.sup.1 may
be introduced on H--R.sup.1--H by heating a reagent of formula
H--R.sup.1--H, which can either be mono-protected by a suitable
protecting group (i.e. PG-R.sup.1--H), or not if it is symmetrical,
with sulfamide in a suitable solvent, such as dioxane, under
microwave irradiation. The protecting group may then be removed by
methods known in the art, for example by reaction with TFA in
dichloromethane when the protecting group is a Boc-protecting
group, leading to the monosulfamide derivatized R.sup.1 chain.
Method D
[0104] Compounds of formula A-3 or A-4 may undergo functional group
manipulation, such as alkylation or reductive amination, before PG
removal of compounds A-3 and/or reaction leading to the sulfamide
A-4.
Method E
##STR00046## ##STR00047##
[0106] The ester bearing the Ra group of compounds A-1 (Ra being
for example a tert-butyl group and Rb a methyl group) may be
hydrolyzed as described above, in acidic conditions, using for
instance TFA in a suitable solvent like DCM, to yield the
carboxylic acid derivative E-2.
[0107] Reaction of compounds E-2 with the sulfamide moiety
introduced on a mono-protected bivalent chain R1, may lead to the
acyl sulfamide compounds E-3, using the conditions described for
the last step of method A. Preferably the coupling agent used to
activate the carboxylic acid group may be CDI, in a suitable
solvent like acetonitrile or THF, under heating conditions.
Addition of the sulfamide chain in the presence of a base such as
DBU may subsequently lead to compounds E-3. PG is a suitable amine
protecting group, chosen from the ones known in the art.
Preferably, within method E, PG is a Boc-protecting group.
[0108] Removal of the protecting group PG of compounds E-3
following methods known in the art may lead to compounds E-4. These
methods include the reaction of compounds E-3 with TFA in a
suitable solvent such as DCM, when PG is a Boc-protecting group.
The ester function of compounds E-4 (Rb is a methyl group) may then
be hydrolyzed, using conditions known in the art, and including the
saponification in basic media as described above, leading to
compounds E-5.
[0109] Alternatively, compounds E-3 may undergo the saponification
reaction in basic media to hydrolyze the ester bearing Rb, prior to
the removal of the amine protecting group using the conditions
described above, and leading to compounds E-5.
[0110] Compounds (I) may be obtained by macrocyclisation of
compounds E-5 by forming the intramolecular amide bond, in the
presence of coupling agents, as described in method A. Preferably
this amide formation step may be performed under high dilution
conditions.
Method F
##STR00048## ##STR00049##
[0112] Compounds F-3 may be obtained by an amide forming reaction,
starting from compounds A-2 and an alkenylamine, as described for
the second step of method A. Subsequent ester hydrolysis under
basic or acidic conditions as described previously may lead to
compounds F-4. The acylsulfamide bond may then be formed using the
method described for the last step of method A, using an alkenyl
sulfamide compound and leading to compounds F-5.
[0113] Alternatively, the acylsulfamide group may be introduced on
a compound of formula E-2, prior to the hydrolysis of the ester
bearing the Rb group and coupling of the obtained carboxylic acid
with an alkenamine as described above, leading to compound F-5.
[0114] Formation of the macrocycle, i.e. compound of formula F-6,
which is a compound of formula (I) bearing the following bivalent
chain as R.sup.1:
##STR00050##
can be carried out via an olefin metathesis reaction in the
presence of a suitable metal catalyst such as e.g. the Ru-based
catalyst reported by Miller, S. J., Blackwell, H. E., Grubbs, R. H.
J. Am. Chem. Soc. 118, (1996), 9606-9614; Kingsbury, J. S.,
Harrity, J. P. A., Bonitatebus, P. J., Hoveyda, A. H., J. Am. Chem.
Soc. 121, (1999), 791-799; and Huang et al., J. Am. Chem. Soc. 121,
(1999), 2674-2678; for example a Hoveyda-Grubbs catalyst.
[0115] Air-stable ruthenium catalysts such as
bis(tricyclohexylphosphine)-3-phenyl-1H-inden-1-ylidene ruthenium
chloride (Neolyst M1.RTM.) or
bis(tricyclohexylphosphine)-[(phenylthio)methylene]ruthenium (IV)
dichloride can be used. Other catalysts that can be used are Grubbs
first and second generation catalysts, i.e.
benzylidene-bis(tricyclo-hexylphosphine)dichlororuthenium and
(1,3-bis-(2,4,6-trimethylphenyl)-2-imidazolidinylidene)dichloro(phenylmet-
hylene)(tricyclohexylphosphine)ruthenium, respectively. Of
particular interest are the Hoveyda-Grubbs first and second
generation catalysts, which are
dichloro(o-isopropoxyphenylmethylene)(tricyclohexylphosphine)-r-
uthenium(II) and
1,3-bis-(2,4,6-trimethylphenyl)-2-imidazolidinylidene)dichloro-(o-isoprop-
oxyphenylmethylene)ruthenium respectively. Also, other catalysts
containing other transition metals such as Mo can be used for this
reaction.
[0116] The metathesis reactions may be conducted in a suitable
solvent such as for example ethers, e.g. THF, dioxane; halogenated
hydrocarbons, e.g. dichloromethane, CHCl.sub.3, 1,2-dichloroethane
and the like, hydrocarbons, e.g. toluene. These reactions are
conducted at increased temperatures under nitrogen atmosphere.
[0117] Compounds of formula (I) or any subgroup thereof or any
subgroups thereof may be converted into each other following
art-known functional group transformation reactions. For example,
amino groups may be N-alkylated, nitro groups reduced to amino
groups, a halo atom may be exchanged for another halo.
[0118] Compounds of formula F-6 may be submitted to catalytic
hydrogenation, using for example Pd/C as a catalyst, in a suitable
solvent such as methanol, ethanol, THF, acetic acid or a mixture
thereof, to yield compounds of formula F-7, where the alkene of the
bivalent chain R1 is reduced to the corresponding alkane. Compounds
of formula F-6 belonging to the group of compounds of formula (II)
may lead to compounds F-7 having the structure of compounds of
formula (IV) after this hydrogenation step. More generally, a
compound of formula (II) may be transformed to a compound of
formula (IV) by catalytic hydrogenation as shown below.
##STR00051##
[0119] The compounds of formula (I) may be converted to the
corresponding N-oxide forms following art-known procedures for
converting a trivalent nitrogen into its N-oxide form. Said
N-oxidation reaction may generally be carried out by reacting the
starting material of formula (I) with an appropriate organic or
inorganic peroxide. Appropriate inorganic peroxides comprise, for
example, hydrogen peroxide, alkali metal or earth alkaline metal
peroxides, e.g. sodium peroxide, potassium peroxide; appropriate
organic peroxides may comprise peroxy acids such as, for example,
benzenecarboperoxoic acid or halo substituted benzenecarboperoxoic
acid, e.g. 3-chlorobenzenecarboperoxoic acid, peroxoalkanoic acids,
e.g. peroxoacetic acid, alkylhydroperoxides, e.g. tert-butyl
hydroperoxide. Suitable solvents are, for example, water, lower
alcohols, e.g. ethanol and the like, hydrocarbons, e.g. toluene,
ketones, e.g. 2-butanone, halogenated hydrocarbons, e.g.
dichloromethane, and mixtures of such solvents.
[0120] Pure stereochemically isomeric forms of the compounds of
formula (I) may be obtained by the application of art-known
procedures. Diastereomers may be separated by physical methods such
as selective crystallization and chromatographic techniques, e.g.,
counter-current distribution, liquid chromatography and the
like.
[0121] The compounds of formula (I) may be obtained as racemic
mixtures of enantiomers which can be separated from one another
following art-known resolution procedures. The racemic compounds of
formula (I), which are sufficiently basic or acidic may be
converted into the corresponding diastereomeric salt forms by
reaction with a suitable chiral acid, respectively chiral base.
Said diastereomeric salt forms are subsequently separated, for
example, by selective or fractional crystallization and the
enantiomers are liberated therefrom by alkali or acid. An
alternative manner of separating the enantiomeric forms of the
compounds of formula (I) involves liquid chromatography, in
particular liquid chromatography using a chiral stationary phase.
Said pure stereochemically isomeric forms may also be derived from
the corresponding pure stereochemically isomeric forms of the
appropriate starting materials, provided that the reaction occurs
stereospecifically. Preferably if a specific stereoisomer is
desired, said compound may be synthesized by stereospecific methods
of preparation. These methods may advantageously employ
enantiomerically pure starting materials.
[0122] Method G describes the synthesis of enantiomerically pure
starting materials A-2, belonging to the groups of compounds (III)
and (IV).
Method G
##STR00052##
[0124] A racemic mixture A-2 may be reacted with a chiral
auxiliary, such as (S)-4-benzyl-2-oxazolidinone, after having being
transformed to its acylchloride using methods known in the art,
such as reaction of A-2 with oxalyl chloride in a suitable solvent
like THF, in the presence of a catalytic amount of DMF. The acid
chloride may then be reacted with the anion of
(S)-4-benzyl-2-oxazolidinone formed by the reaction with a strong
base, such as butyl lithium, in a suitable solvent such as THF, at
low temperatures, typically -78.degree. C., and under an inert
atmosphere, leading to the diastereoisomers G1 and G2, which can be
isolated by methods known in the art, such as chromatography on
silica gel.
[0125] Removal of the chiral auxiliary from each of the
diastereoisomers G1 and G2 may then be performed with a base such
as NaOH in a suitable solvent, such as methanol, water, THF,
leading to the enantiomerically pure compounds A-2' and A-2''.
Using these enantiomerically pure starting materials may lead to
enantiomerically pure compounds of formula (I) bearing one
stereocenter, such as compounds of formula (IIIA), (IIIB).
[0126] Pure stereochemically isomeric forms of the compounds of
formula (I) or any subgroups thereof may be obtained by the
application of art-known procedures. Diastereomers may be separated
by physical methods such as selective crystallization and
chromatographic techniques, e.g., counter-current distribution,
liquid chromatography and the like.
[0127] The compounds of formula (I) or any subgroups thereof may be
obtained as racemic mixtures of enantiomers, which can be separated
from one another following art-known resolution procedures. The
racemic compounds of formula (I) or any subgroups thereof, which
are sufficiently basic or acidic may be converted into the
corresponding diastereomeric salt forms by reaction with a suitable
chiral acid, respectively chiral base. Said diastereomeric salt
forms are subsequently separated, for example, by selective or
fractional crystallization and the enantiomers are liberated
therefrom by alkali or acid. An alternative manner of separating
the enantiomeric forms of the compounds of formula (I) or any
subgroups thereof involves liquid chromatography, in particular
liquid chromatography using a chiral stationary phase. Said pure
stereochemically isomeric forms may also be derived from the
corresponding pure stereochemically isomeric forms of the
appropriate starting materials, provided that the reaction occurs
stereospecifically. Preferably if a specific stereoisomer is
desired, said compound may be synthesized by stereospecific methods
of preparation. These methods may advantageously employ
enantiomerically pure starting materials.
[0128] In a further aspect, the present invention concerns a
pharmaceutical composition comprising a therapeutically effective
amount of a compound of formula (I) or any subgroups thereof, as
specified herein, and a pharmaceutically acceptable carrier. A
therapeutically effective amount in this context is an amount
sufficient to prophylactically act against, to stabilize or to
reduce viral infection, and in particular HCV viral infection, in
infected subjects or subjects being at risk of being infected. In
still a further aspect, this invention relates to a process of
preparing a pharmaceutical composition as specified herein, which
comprises intimately mixing a pharmaceutically acceptable carrier
with a therapeutically effective amount of a compound of formula
(I) or any subgroups thereof, as specified herein.
[0129] Therefore, according to an embodiment of the present
invention, the compounds of formula (I) or any subgroup thereof may
be formulated into various pharmaceutical forms for administration
purposes. It is understood that all compositions usually employed
for systemically administering drugs are included as appropriate
compositions. To prepare the pharmaceutical compositions of this
invention, an effective amount of the particular compound,
optionally in salt form or a metal complex, as the active
ingredient is combined in intimate admixture with a
pharmaceutically acceptable carrier, which carrier may take a wide
variety of forms depending on the form of preparation desired for
administration. These pharmaceutical compositions are desirable in
unitary dosage form suitable, particularly, for administration
orally, rectally, percutaneously, or by parenteral injection. For
example, in preparing the compositions in oral dosage form, any of
the usual pharmaceutical media may be employed such as, for
example, water, glycols, oils, alcohols and the like in the case of
oral liquid preparations such as suspensions, syrups, elixirs,
emulsions and solutions; or solid carriers such as starches,
sugars, kaolin, lubricants, binders, disintegrating agents and the
like in the case of powders, pills, capsules, and tablets. Because
of their ease in administration, tablets and capsules represent the
most advantageous oral dosage unit forms, in which case solid
pharmaceutical carriers are obviously employed. For parenteral
compositions, the carrier will usually comprise sterile water, at
least in large part, though other ingredients, for example, to aid
solubility, may be included. Injectable solutions, for example, may
be prepared in which the carrier comprises saline solution, glucose
solution or a mixture of saline and glucose solution. Injectable
suspensions may also be prepared in which case appropriate liquid
carriers, suspending agents and the like may be employed. Also
included are solid form preparations that are intended to be
converted, shortly before use, to liquid form preparations. In the
compositions suitable for percutaneous administration, the carrier
optionally comprises a penetration enhancing agent and/or a
suitable wetting agent, optionally combined with suitable additives
of any nature in minor proportions, which additives do not
introduce a significant deleterious effect on the skin.
[0130] The compounds of the present invention may also be
administered via oral inhalation or insufflation by means of
methods and formulations employed in the art for administration via
this way. Thus, in general the compounds of the present invention
may be administered to the lungs in the form of a solution, a
suspension or a dry powder, a solution being preferred. Any system
developed for the delivery of solutions, suspensions or dry powders
via oral inhalation or insufflation are suitable for the
administration of the present compounds.
[0131] Thus, the present invention also provides a pharmaceutical
composition adapted for administration by inhalation or
insufflation through the mouth comprising a compound of formula (I)
or any subgroups thereof and a pharmaceutically acceptable carrier.
Preferably, the compounds of the present invention are administered
via inhalation of a solution in nebulized or aerosolized doses.
[0132] It is especially advantageous to formulate the
aforementioned pharmaceutical compositions in unit dosage form for
ease of administration and uniformity of dosage. Unit dosage form
as used herein refers to physically discrete units suitable as
unitary dosages, each unit containing a predetermined quantity of
active ingredient calculated to produce the desired therapeutic
effect in association with the required pharmaceutical carrier.
Examples of such unit dosage forms are tablets (including scored or
coated tablets), capsules, pills, suppositories, powder packets,
wafers, injectable solutions or suspensions and the like, and
segregated multiples thereof.
[0133] The compounds of formula (I) and any subgroup thereof show
antiviral properties. Viral infections and their associated
diseases treatable using the compounds and methods of the present
invention include those infections brought on by HCV and other
pathogenic flaviviruses such as Yellow fever, Dengue fever (types
1-4), St. Louis encephalitis, Japanese encephalitis, Murray valley
encephalitis, West Nile virus and Kunjin virus. The diseases
associated with HCV include progressive liver fibrosis,
inflammation and necrosis leading to cirrhosis, end-stage liver
disease, and HCC; and for the other pathogenic flaviviruses the
diseases include yellow fever, dengue fever, hemorrhagic fever and
encephalitis.
[0134] However, compounds of the invention may also be attractive
due to the fact that they lack activity against other viruses, in
particular against HIV. HIV infected patients often suffer from
co-infections such as HCV. Treatment of such patients with an HCV
inhibitor that also inhibits HIV may lead to the emergence of
resistant HIV strains.
[0135] Due to their antiviral properties, particularly their
anti-HCV properties, the compounds of formula (I) or any subgroup
thereof, including stereochemically isomeric forms, and their
N-oxides, quaternary amines, metal complexes, salts, hydrates and
solvates, are useful in the treatment of individuals experiencing a
viral infection, particularly a HCV infection, and for the
prophylaxis of these infections. In general, the compounds of the
present invention may be useful in the treatment of warm-blooded
animals infected with viruses, in particular flaviviruses such as
HCV.
[0136] The compounds of the present invention or any subgroup
thereof may therefore be used as medicines. Said use as a medicine
or method of treatment comprises the systemic administration to
virally infected subjects or to subjects susceptible to viral
infections of an amount effective to combat the conditions
associated with the viral infection, in particular the HCV
infection.
[0137] The present invention also relates to the use of the present
compounds or any subgroup thereof in the manufacture of a
medicament for the treatment or the prevention of viral infections,
particularly HCV infection.
[0138] The present invention furthermore relates to a method of
treating a warm-blooded animal infected by a virus, or being at
risk of infection by a virus, in particular by HCV, said method
comprising the administration of an anti-virally effective amount
of a compound of formula (I), or any subgroups thereof, as
specified herein.
[0139] The present invention also concerns combinations of a
compound of formula (I) or any subgroup thereof, as specified
herein with other anti-HCV agents. In an embodiment, the invention
concerns combination of a compound of Formula (I) or any subgroup
thereof with at least one anti-HCV agent. In a particular
embodiment, the invention concerns combination of a compound of
Formula (I) or any subgroup thereof with at least two anti-HCV
agents. In a particular embodiment, the invention concerns
combination of a compound of Formula (I) or any subgroup thereof
with at least three anti-HCV agents. In a particular embodiment,
the invention concerns combination of a compound of Formula (I) or
any subgroup thereof with at least four anti-HCV agents.
[0140] The combination of previously known anti-HCV compound, such
as interferon-.alpha. (IFN-.alpha.), pegylated interferon-.alpha.,
ribavirin or a combination thereof, and, a compound of formula (I)
or any subgroup thereof can be used as a medicine in a combination
therapy. In an embodiment, the term "combination therapy" relates
to a product containing mandatory (a) a compound of formula (I),
and (b) at least one other anti-HCV compound, as a combined
preparation for simultaneous, separate or sequential use in
treatment of HCV infections, in particular, in the treatment of
infections with HCV.
[0141] Anti-HCV compounds encompass agents selected from HCV
polymerase inhibitors, R-7128, MK-0608, VCH759, PF-868554, GS9190,
NM283, valopicitabine, PSI-6130, XTL-2125, NM-107, R7128 (R4048),
GSK625433, R803, R-1626, BILB-1941, HCV-796, JTK-109 and JTK-003,
ANA-598, IDX-184, MK-3281, MK-1220, benzimidazole derivatives,
benzo-1,2,4-thiadiazine derivatives, phenylalanine derivatives,
A-831 and A-689; HCV proteases (NS2-NS3 and NS3-NS4A) inhibitors,
the compounds of WO02/18369 (see, e.g., page 273, lines 9-22 and
page 274, line 4 to page 276, line 11), BI-1335, TMC435350, MK7009,
ITMN-191, BILN-2061, VX-950, BILN-2065, BMS-605339, VX-500, SCH
503034; inhibitors of other targets in the HCV life cycle,
including helicase, and metalloprotease inhibitors, ISIS-14803;
immunomodulatory agents such as, .alpha.-, .beta.-, and
.gamma.-interferons such as rIFN-.alpha. 2b, rIFN-.alpha. 2ba,
consensus IFN-.alpha.(infergen), feron, reaferon, intermax .alpha.,
rIFN-.beta., infergen+actimmune, IFN-omega with DUROS, albuferon,
locteron, Rebif, Oral IFN-.alpha., IFN-.alpha.2b XL, AVI-005,
pegylated-infergen, pegylated derivatized interferon-.alpha.
compounds such as pegylated rIFN-.alpha. 2b, pegylated rIFN-.alpha.
2a, pegylated IFN-.beta., compounds that stimulate the synthesis of
interferon in cells, interleukins, Toll like receptor (TLR)
agonists, compounds that enhance the development of type 1 helper T
cell response, and thymosin; other antiviral agents such as
ribavirin, ribavirin analogs such as rebetol, copegus and
viramidine (taribavirin), amantadine, and telbivudine, inhibitors
of internal ribosome entry, alpha-glucosidase 1 inhibitors such as
MX-3253 (celgosivir) and UT-231B, hepatoprotectants such as
IDN-6556, ME-3738, LB-84451 and MitoQ, broad-spectrum viral
inhibitors, such as IMPDH inhibitors (e.g., compounds of U.S. Pat.
No. 5,807,876, U.S. Pat. No. 6,498,178, U.S. Pat. No. 6,344,465,
U.S. Pat. No. 6,054,472, WO97/40028, WO98/40381, WO00/56331,
mycophenolic acid and derivatives thereof, and including, but not
limited to VX-497, VX-148, and/or VX-944); and other drugs for
treating HCV such as zadaxin, nitazoxanide, BIVN-401 (virostat),
PYN-17 (altirex), KPE02003002, actilon (CPG-10101), KRN-7000,
civacir, G1-5005, ANA-975, XTL-6865, ANA-971, NOV-205, tarvacin,
EHC-18, NIM811, DEBIO-025, VGX-410C, EMZ-702, AVI 4065,
Bavituximab, and Oglufanide; or combinations of any of the
above.
[0142] Thus, to combat or treat HCV infections, the compounds of
formula (I) or any subgroups thereof may be co-administered in
combination with for instance, interferon-.alpha. (IFN-.alpha.),
pegylated interferon-.alpha., ribavirin or a combination thereof,
as well as therapeutics based on antibodies targeted against HCV
epitopes, small interfering RNA (si RNA), ribozymes, DNAzymes,
antisense RNA, small molecule antagonists of for instance NS3
protease, NS3 helicase and NS5B polymerase.
[0143] The combinations of the present invention may be used as
medicaments. Accordingly, the present invention relates to the use
of a compound of formula (I) or any subgroup thereof as defined
above for the manufacture of a medicament useful for inhibiting HCV
activity in a mammal infected with HCV viruses, wherein said
medicament is used in a combination therapy, said combination
therapy preferably comprising a compound of formula (I) and at
least one other HCV inhibitory compound, e.g. IFN-.alpha.,
pegylated IFN-.alpha., ribavirin or a combination thereof.
[0144] Furthermore, it is known that a large percentage of patients
infected with human immunodeficiency virus 1 (HIV) are also
infected with HCV, i.e. they are HCV/HIV co-infected. HIV infection
appears to adversely affect all stages of HCV infection, leading to
increased viral persistence and accelerated progression of
HCV-related liver disease. In turn, HCV infection may affect the
management of HIV infection, increasing the incidence of liver
toxicity caused by antiviral medications.
[0145] The present invention therefore also concerns combinations
of a compound of Formula (I) or any subgroup thereof with anti-HIV
agents. Also, the combination of one or more additional anti-HIV
compounds and a compound of Formula (I) or any subgroups thereof
can be used as a medicine. In particular, said combination can be
used for inhibition HCV and HIV replication.
[0146] The term "combination therapy" also encompasses a product
comprising (a) a compound of Formula (I) or any subgroup thereof,
and (b) at least one anti-HIV compound, and (c) optionally at least
one other anti-HCV compound, as a combined preparation for
simultaneous, separate or sequential use in treatment of HCV and
HIV infections, in particular, in the treatment of infections with
HCV and HIV, or for preventing or treating conditions associated
with HCV and HIV.
[0147] Thus, the present invention also relates to a product
containing (a) at least one compound of Formula (I) or any subgroup
thereof, and (b) one or more additional anti-HIV compounds, as a
combined preparation for simultaneous, separate or sequential use
in anti-HCV and anti-HIV treatment. The different drugs may be
combined in a single preparation together with pharmaceutically
acceptable carriers. Said anti-HIV compounds may be any known
antiretroviral compounds such as suramine, pentamidine,
thymopentin, castanospermine, dextran (dextran sulfate),
foscarnet-sodium (trisodium phosphono formate); nucleoside reverse
transcriptase inhibitors (NRTIs), e.g. zidovudine (AZT), didanosine
(ddI), zalcitabine (ddC), lamivudine (3TC), stavudine (d4T),
emtricitabine (FTC), abacavir (ABC), amdoxovir (DAPD), elvucitabine
(ACH-126,443), AVX 754 ((-)-dOTC), fozivudine tidoxil (FZT),
phosphazide, HDP-990003, KP-1461, MIV-210, racivir (PSI-5004),
UC-781 and the like; non-nucleoside reverse transcriptase
inhibitors (NNRTIs) such as delavirdine (DLV), efavirenz (EFV),
nevirapine (NVP), dapivirine (TMC 120), etravirine (TMC125),
rilpivirine (TMC278), DPC-082, (+)-Calanolide A, BILR-355, and the
like; nucleotide reverse transcriptase inhibitors (NtRTIs), e.g.
tenofovir ((R)--PMPA) and tenofovir disoproxil fumarate (TDF), and
the like; nucleotide-competing reverse transcriptase inhibitors
(NcRTIs), e.g. NcRTI-1 and the like; inhibitors of trans-activating
proteins, such as TAT-inhibitors, e.g. RO-5-3335, BI-201, and the
like; REV inhibitors; protease inhibitors e.g. ritonavir (RTV),
saquinavir (SQV), lopinavir (ABT-378 or LPV), indinavir (IDV),
amprenavir (VX-478), TMC126, nelfinavir (AG-1343), atazanavir (BMS
232,632), darunavir (TMC114), fosamprenavir (GW433908 or VX-175),
brecanavir (GW-640385, VX-385), P-1946, PL-337, PL-100, tipranavir
(PNU-140690), AG-1859, AG-1776, Ro-0334649 and the like; entry
inhibitors, which comprise fusion inhibitors (e.g. enfuvirtide
(T-20)), attachment inhibitors and co-receptor inhibitors, the
latter comprise the CCR5 antagonists (e.g. ancriviroc, CCR5 mAb004,
maraviroc (UK-427,857), PRO-140, TAK-220, TAK-652, vicriviroc
(SCH-D, SCH-417,690)) and CXR4 antagonists (e.g. AMD-070,
KRH-27315), examples of entry inhibitors are PRO-542, TNX-355,
BMS-488043, BlockAide/CR.TM., FP 21399, hNM01, nonakine, VGV-1; a
maturation inhibitor for example is PA-457; inhibitors of the viral
integrase e.g. raltegravir (MK-0518), elvitegravir (JTK-303,
GS-9137), BMS-538158; ribozymes; immunomodulators; monoclonal
antibodies; gene therapy; vaccines; siRNAs; antisense RNAs;
microbicides; Zinc-finger inhibitors.
[0148] Therefore, HCV infected patients also suffering from
conditions associated with HIV or even other pathogenic
retroviruses, such as AIDS, AIDS-related complex (ARC), progressive
generalized lymphadenopathy (PGL), as well as chronic CNS diseases
caused by retroviruses, such as, for example HIV mediated dementia
and multiple sclerosis, can conveniently be treated with the
present composition.
[0149] The compositions may be formulated into suitable
pharmaceutical dosage forms such as the dosage forms described
above. Each of the active ingredients may be formulated separately
and the formulations may be co-administered or one formulation
containing both and if desired further active ingredients may be
provided.
[0150] As used herein, the term "composition" is intended to
encompass a product comprising the specified ingredients, as well
as any product that results, directly or indirectly, from the
combination of the specified ingredients.
[0151] The term "therapeutically effective amount" as used herein
means that amount of active compound or component or pharmaceutical
agent that elicits the biological or medicinal response in a
tissue, system, animal or human that is being sought, in the light
of the present invention, by a researcher, veterinarian, medical
doctor or other clinician, which includes alleviation of the
symptoms of the disease being treated. Since the instant invention
refers as well to combinations comprising two or more agents, the
"therapeutically effective amount" in the context of combinations
is also that amount of the agents taken together so that the
combined effect elicits the desired biological or medicinal
response. For example, the therapeutically effective amount of a
composition comprising (a) the compound of formula (I) and (b)
another anti-HCV agent, would be the amount of the compound of
formula (I) and the amount of the other anti-HCV agent that when
taken together have a combined effect that is therapeutically
effective.
[0152] In general, it is contemplated that an antiviral effective
daily amount would be from 0.01 mg/kg to 500 mg/kg body weight,
more preferably from 0.1 mg/kg to 50 mg/kg body weight. It may be
appropriate to administer the required dose as two, three, four or
more sub-doses at appropriate intervals throughout the day. Said
sub-doses may be formulated as unit dosage forms, for example,
containing 1 to 1000 mg, and in particular 5 to 200 mg of active
ingredient per unit dosage form.
[0153] The exact dosage and frequency of administration depends on
the particular compound of formula (I) used, the particular
condition being treated, the severity of the condition being
treated, the age, weight, sex, extent of disorder and general
physical condition of the particular patient as well as other
medication the individual may be taking, as is well known to those
skilled in the art. Furthermore, it is evident that said effective
daily amount may be lowered or increased depending on the response
of the treated subject and/or depending on the evaluation of the
physician prescribing the compounds of the instant invention. The
effective daily amount ranges mentioned hereinabove are therefore
only guidelines.
[0154] In one embodiment of the present invention there is provided
an article of manufacture comprising a composition effective to
treat an HCV infection or to inhibit the NS5B polymerase of HCV;
and packaging material comprising a label which indicates that the
composition can be used to treat infection by the hepatitis C
virus; wherein the composition comprises a compound of the formula
(I) or any subgroup thereof, or the combination as described
herein.
[0155] Another embodiment of the present invention concerns a kit
or container comprising a compound of the formula (I) or any
subgroup thereof, in an amount effective for use as a standard or
reagent in a test or assay for determining the ability of potential
pharmaceuticals to inhibit HCV NS5B polymerase, HCV growth, or
both. This aspect of the invention may find its use in
pharmaceutical research programs.
[0156] The compounds and combinations of the present invention can
be used in high-throughput target-analyte assays such as those for
measuring the efficacy of said combination in HCV treatment.
EXAMPLES
[0157] The following examples are intended to illustrate the
present invention and not to limit it thereto. Unless otherwise
indicated, purification of the synthesized compounds by column
chromatography or flash chromatography is performed on a silica gel
column.
Example 1
Synthesis of Compound 1
##STR00053##
[0158] Step 1
##STR00054##
[0160] A solution of NaOH (6.38 g) in 25 mL of water was added to a
stirred solution of 1a (10-tert-butyl 6-methyl
13-cyclohexyl-3-methoxy-7H-indolo[2,1-a][2]benzazepine-6,10-dicarboxylate-
, synthesized as described in US 2007270406 A1) in THF (100 mL) and
MeOH (150 mL). After 1 hour the reaction was concentrated under
reduced pressure, then diluted with ice-cold water (150 mL). The pH
of the resulting solution was adjusted to 6 with acetic acid
(AcOH). The precipitate was collected by filtration, washed with
water and dried under vacuum to give 1.90 g (98%) of 1b as a
yellowish powder: m/z=488 (M+H).sup.+
Step 2
##STR00055##
[0162] HATU (1.17 g, 3.08 mmol) was added under nitrogen to a
stirred solution of 1b (1.00 g, 2.05 mmol), DIPEA (1.07 mL, 6.15
mmol) and 2,2'-oxybis(N-methylethanamine) (1.08 g, 8.20 mmol) in 30
mL of dry THF. After 1 h, the reaction mixture was quenched with
water (100 mL) and extracted with ethyl acetate (EtOAc). The
organic layer was successively dried (Na.sub.2SO.sub.4), filtered
and evaporated. The residue was triturated in water, filtered and
dried to give 1.15 g (93%) the target compound 1c as a yellowish
powder: m/z=602 (M+H).sup.+
Step 3
##STR00056##
[0164] A solution of 1c (1.15 g, 1.91 mmol) and sulfamide (1.84 g,
19.1 mmol) in dioxane (10 mL) was heated at 100.degree. C. in a
microwave oven for 20 minutes. The reaction mixture was cooled down
to room temperature, then evaporated under vacuum. The residue was
triturated in water, filtered and washed with water. The powder was
reconstituted in EtOAc, dried (Na.sub.2SO.sub.4) and evaporated to
give 1.15 g (88%) of the desired product 1d as a yellowish powder:
m/z=681 (M+H).sup.+
Step 4
##STR00057##
[0166] TFA (3.0 g, 26.3 mmol) was added to a solution of 1d (1.15
g, 1.70 mmol) in dichloromethane (3 mL). After 1 h, the reaction
mixture was concentrated under vacuum. The residue was triturated
in ether, filtered and washed with ether, then purified by
chromatography (gradient EtOAc to EtOAc/EtOH, 9:1) to give 802 mg
(76%) of the desired product 1e: m/z=625 (M+H).sup.30
Step 5
##STR00058##
[0168] Carbonyldiimidazole (389 mg, 2.40 mmol) was added to a
stirred solution of 1e (500 mg, 0.80 mmol) in dry THF (3 mL). The
reaction mixture was stirred at room temperature for 1 h: complete
conversion to intermediate if was observed. The resulting solution
was evaporated, then the residue was purified by flash
chromatography (gradient EtOAc to CH.sub.3CN 1:0 to 0:1) to give
550 mg of the target product 1f which was used as such in the next
step: m/z=675 (M+H).sup.+
Step 6
##STR00059##
[0170] DBU (244 mg, 0.32 mmol) was added to a solution of 1f (550
mg) in acetonitrile (25 mL). The reaction mixture was stirred
overnight at room temperature, then concentrated under reduced
pressure. The residue was dissolved in water (30 mL) and the pH of
the resulting solution was adjusted to 5. The precipitate was
collected by filtration, washed with water and dried.
Recrystallization from ethanol followed by a purification by column
chromatography (gradient EtOAc to EtOAc/EtOH 9:1) provided 380 mg
(78%) of the title product 1 as a white powder: m/z=607 (M+H)+,
.sup.1H NMR (DMSO-d.sub.6) .delta. 1.15 (m, 1H), 1.40 (m, 3H), 1.71
(m, 2H), 1.88 (m, 1H), 2.01 (m, 3H), 2.56 (m, 3H), 2.77 (m, 1H),
2.99 (s, 3H), 3.26 (m, 2H), 3.50-3.71 (m, 6H), 3.87 (s, 3H), 4.44
(d, J=14.1 Hz, 1H), 5.09 (d, J=15.0 Hz, 1H), 6.95 (s, 1H), 7.13 (s,
1H), 7.19 (d, J=8.6 Hz, 1H), 7.47 (d, J=8.0 Hz, 1H), 7.54 (d, J=8.3
Hz, 1H), 7.88 (d, J=7.8 Hz, 1H), 8.33 (s, 1H), 11.40 (s, 1H).
Example 2
Synthesis of Compound 2
##STR00060##
[0172] A solution of 1 (56 mg, 0.092 mmol) in MeOH (15 mL) and THF
(5 mL) was hydrogenated in an H-cube apparatus using a 10% Pd on
Carbon cartridge. Then, solvent was evaporated and the residue was
purified by column chromatography (CH.sub.2Cl.sub.2/CH.sub.3CN,
9:1) to give 23 mg (41%) of the desired product 2 as a white
powder: m/z=609 (M+H).sup.+.
Examples 3 and 4
Synthesis of Compounds 3 and 4
##STR00061##
[0174] The racemic mixture 2 was purified by SFC, using a 6.5
minutes run on a chiral CHIRALCEL OD-H column (250.times.10 mm,
coated on 5 .mu.m silica gel) and 55% methanol/45% CO.sub.2 as
mobile phase, at a flow rate of 10 mL/min and lead to the two pure
enantiomers 3 and 4. Retention times under these conditions were
observed at 4.25 min and 5.54 min.
Example 5
Synthesis of Compound 5
##STR00062##
[0175] Step 1
##STR00063##
[0177] The compound 5a was synthesized in 96% yield from
intermediate 1b and 2-[4-(tert-butyloxy
carbonyl)piperazin-1-yl]ethylamine following the procedure reported
for the synthesis of intermediate 1c: m/z=699 (M+H).sup.+.
Step 2
##STR00064##
[0179] Trifluoroacetic acid (5.00 g, 43.9 mmol) was added to 740 mg
of intermediate 5a. After 1 hour at room temperature, the solvent
was evaporated. The residue was triturated in EtOH/Et.sub.2O,
filtered and dried under high vacuum to give 380 mg (64%) of the
desired product 5b as a yellowish powder: m/z=543 (M+H).sup.+.
Step 3
##STR00065##
[0181] A solution of 5b (380 mg, 0.700 mmol) and sulfamide (673 mg,
7.00 mmol) in dioxane (10 mL) was heated at 100.degree. C. in a
microwave oven for 15 minutes. Then, the reaction mixture was
successively cooled down at room temperature, concentrated under
vacuum, triturated in water and filtered. Purification by column
chromatography (gradient EtOAc/CH.sub.2Cl.sub.2 1:1 to 1:0) gave
210 mg (46%) of the desired product 5c: m/z=622 (M+H).sup.+.
Step 4
##STR00066##
[0183] The title product 5d was synthesized in 11% yield following
the procedure (steps 5 and 6) reported for the synthesis of
compound 1, followed by a recrystallization from ethanol, affording
the desired product as white powder, m/z=604 (M+H).sup.+.
Example 6
Synthesis of Compound 6
##STR00067##
[0184] Step 1
##STR00068##
[0186] To a solution of
N.sub.1-(2-aminoethyl)-N.sub.1-methylethane-1,2-diamine (10.58 g,
90 mmoles) in DCM (350 mL) was added slowly a solution of
2-nitrobenzene-1-sulfonyl chloride dissolved in DCM (50 mL). After
2 h at RT, the reaction mixture (RM) was washed with water, dried
over MgSO.sub.4, filtered and concentrated. The residue was
purified by flash chromatography on silica gel with a gradient of
methanol in DCM (0 to 10%), yielding to 6.9 g of
N-(2-((2-aminoethyl)(methyl)-amino)ethyl)-2-nitrobenzenesulfonamide
6a and 3.9 g of
N,N'-(2,2'-(methylazanediyl)-bis(ethane-2,1-diyl))bis(2-nitrobenzenesulfo-
namide) 6b; m/z (6a)=303 (M+H).sup.+, m/z (6b)=488 (M+H).sup.+.
Step 2
##STR00069##
[0188] To a solution of carboxylic acid 1b (500 mg, 1.025 mmole),
HATU (585 mg, 1.5 eq) and diisopropylethylamine (212 mg, 1.6 eq) in
dry DMF (10 mL) was added
N-(2-((2-aminoethyl)(methyl)amino)ethyl)-2-nitrobenzenesulfonamide
6a (341 mg, 1.1 eq). After 30 minutes at RT, the RM was diluted
with water. The yellow precipitate was filtered off and washed with
water. It was then reconstituted in EtOAc, dried over MgSO.sub.4,
filtered, concentrated and dried under vacuum to give 800 mg of the
desired product
13-Cyclohexyl-3-methoxy-6-(2-{methyl-[2-(2-nitro-benzenesulfonylamino)-et-
hyl]-amino}-ethylcarbamoyl)-7H-benzo[3,4]azepino[1,2-a]indole-10-carboxyli-
c acid tert-butyl ester 6c as a yellow powder; m/z=772
(M+H).sup.+.
Step 3
##STR00070##
[0190] To a solution of
13-cyclohexyl-3-methoxy-6-(2-{methyl-[2-(2-nitro-benzenesulfonyl-amino)-e-
thyl]-amino}-ethylcarbamoyl)-7H-benzo[3,4]azepino[1,2-a]indole-10-carboxyl-
ic acid tert-butyl ester 6c (650 mg, 0.842 mmole) and cesium
carbonate (1.646-g, 6 eq) in dry DMF (10 mL) was added slowly a
solution of methyl iodide (122 mg, 1.02 mmole) in dry DMF (2 mL).
After stirring for 1 h at RT, the RM was diluted with water and
extracted with EtOAc. The organic layer was then washed with water,
dried over MgSO.sub.4, filtered and concentrated. The residue was
purified by flash chromatography on silica gel, using a gradient of
EtOAc in DCM (0 to 100%), yielding to 550 mg (83% yield) of the
desired product
13-Cyclohexyl-3-methoxy-6-[2-(methyl-{2-[methyl-(2-nitro-benzenesulfonyl)-
-amino]-ethyl}-amino)-ethylcarbamoyl]-7H-benzo[3,4]azepino[1,2-a]indole-10-
-carboxylic acid tert-butyl ester 6d as a yellow solid; m/z=786
(M+H).sup.+.
Step 4
##STR00071##
[0192] A mixture of
13-cyclohexyl-3-methoxy-6-[2-(methyl-{2-[methyl-(2-nitro-benzene-sulfonyl-
)amino]-ethyl}-amino)-ethylcarbamoyl]-7H-benzo[3,4]azepino[1,2-a]indole-10-
-carboxylic acid tert-butyl ester 6d (380 mg, 0.483 mmole), cesium
carbonate (315 mg, 2 eq) and thiophenol (107 mg, 2 eq) in DMF (5
mL) was stirred at RT overnight. Cesium carbonate (315 mg, 2 eq)
and thiophenol (107 mg, 2 eq) were then added to the RM and the RM
was stirred for 1 h. Upon completion of the reaction, the RM was
filtered and loaded on a cartridge containing a SCX-resin,
pre-washed with DCM. After rinsing the cartridge with DCM (several
times, until a colorless fraction was obtained) the product was
eluted with NH.sub.3 in MeOH, yielding to 240 mg of the desired
product
13-cyclohexyl-3-methoxy-6-{2-[methyl-(2-methylamino-ethyl)-amino]-ethylca-
rbamoyl}-7H-benzo[3,4]azepino[1,2-a]indole-10-carboxylic acid
tert-butyl ester 6e, which was further purified by preparative
HPLC; m/z=601 (M+H).sup.+.
Step 5
##STR00072##
[0194] The synthesis of product 6f was performed using the
procedure described for the synthesis of compound 1d, using
intermediate 6e instead of intermediate 1c, yielding 200 mg (50%)
of the target product; m/z=680 (M+H).sup.+.
Step 6
##STR00073##
[0196] The synthesis of product 6g was performed using the
procedure described for the synthesis of compound 1e, using
intermediate 6f instead of intermediate 1d, yielding 187 mg
(quantitative yield) of the target product; m/z=624
(M+H).sup.+.
Step 7
##STR00074##
[0198] The synthesis of product 6 was performed using the procedure
described for the synthesis of compound 1, using intermediate 6g
instead of intermediate 1e, yielding 43 mg (22% yield) of the
target product; m/z=606 (M+H).sup.+.
Example 7
Synthesis of Compound 7
##STR00075##
[0199] Step 1
##STR00076##
[0201] To a solution of
N,N'-(2,2'-(methylazanediyl)bis(ethane-2,1-diyl))bis(2-nitrobenzene-sulfo-
namide) 6b (3 g, 6.15 mmoles) in dry DMF (50 mL) was added portion
wise sodium hydride (738 mg, 3 eq, 60% in mineral oil) at 0.degree.
C. After 20 minutes, a solution of methyl iodide dissolved in dry
DMF (5 mL) was added slowly to the RM. After stirring for 1 h at
RT, the RM was quenched with water and extracted with EtOAc. The
organic layer was washed with water, dried over MgSO.sub.4,
filtered and concentrated. Purification by flash chromatography
with a gradient of EtOAc in DCM (20 to 80%) afforded 1.94 g (61%)
of the desired product
N,N-(2,2'-(methylazanediyl)bis(ethane-2,1-diyl))bis(N-methyl-2-nitrobenze-
nesulfonamide) 7a; m/z=516 (M+H).sup.+.
Step 2
##STR00077##
[0203] A mixture of
N,N'-(2,2'-(methylazanediyl)bis(ethane-2,1-diyl))bis(N-methyl-2-nitro-ben-
zenesulfonamide) 7a (1.24 g, 2.405 mmoles), cesium carbonate (2.35
g, 3 eq) and thiophenol (795 mg, 3 eq) in DMF (25 mL) was stirred
at RT during 1 h. Upon completion of the reaction, the RM was
filtered and loaded on a MP-TsOH cartridge, prewashed with DCM.
After rinsing the cartridge with DCM (several times, until a
colorless fraction was obtained) the product was eluted with
NH.sub.3 in MeOH, yielding to 220 mg (63%) of
N.sub.1,N.sub.2-dimethyl-N.sub.1-(2-(methylamino)ethyl)ethane-1,2-diamine
7b, which was used directly in the next step; m/z=146
(M+H).sup.+.
Step 3
##STR00078##
[0205] The synthesis of
13-cyclohexyl-3-methoxy-6-(methyl-{2-[methyl-(2-methylamino-ethyl)-amino]-
-ethyl}-carbamoyl)-7H-benzo[3,4]azepino[1,2-a]indole-10-carboxylic
acid tert-butyl ester 7c was performed following the procedure
reported for the synthesis of compound 1c, using
N.sub.1,N.sub.2-dimethyl-N.sub.1-(2-(methylamino)ethyl)ethane-1,2-diamine
7b instead of Methyl-[2-(2-methylamino-ethoxy)-ethyl]-amine. After
purification by flash chromatography with a gradient of ammonia in
methanol 7M in EtOAc (5 to 15%), 100 mg of the desired product 7c
were obtained as a yellow oil; m/z=615 (M+H).sup.+.
Step 4
##STR00079##
[0207] The synthesis of 7d was performed following the procedure
reported for the synthesis of compound 1d, using
13-cyclohexyl-3-methoxy-6-(methyl-{2-[methyl-(2-methyl-amino-ethyl)-amino-
]-ethyl}-carbamoyl)-7H-benzo[3,4]azepino[1,2-a]indole-10-carboxylic
acid tert-butyl ester 7c instead of
13-cyclohexyl-3-methoxy-6-{methyl-[2-(2-methylamino-ethoxy)-ethyl]-carbam-
oyl}-7H-benzo[3,4]azepino[1,2-a]indole-10-carboxylic acid
tert-butyl ester 1c. After purification by flash chromatography
with a gradient of methanol in EtOAc (0 to 10%), 50 mg of the
desired product 7d were obtained; m/z=694 (M+H).sup.+.
Step 5
##STR00080##
[0209] The synthesis of 7e was performed following the procedure
reported for the synthesis of compound 1e, using intermediate 7d
instead of intermediate 1d; m/z=638 (M+H).sup.+.
Step 6
##STR00081##
[0211] The synthesis of product 7 is being performed using the
procedure described for the synthesis of compound 1, using
intermediate 7e instead of intermediate 1e.
Example 8
Synthesis of Compound 8
##STR00082##
[0212] Step 1
##STR00083##
[0214] The synthesis of 8b was performed following the procedure
reported for the synthesis of compound 1b, using methyl ester 8a
instead of la. The desired product 8b was obtained in 95% yield as
a light yellow solid; m/z=502 (M+H).sup.+.
Step 2
##STR00084##
[0216] At 0.degree. C. and under protective atmosphere, oxalyl
chloride (4.07 ml, 47.4 mmol) was added to a solution of carboxylic
acid 8b (19.83 g, 39.5 mmol) and DMF (5 drops) in tetrahydrofuran
(dry) (100 mL). Upon addition of oxalyl chloride immediate
formation of gas was observed. The reaction was stirred at
0.degree. C. for 1.5 hour. Then, an extra amount of 0.5 eq of
oxalyl chloride was added and the reaction was stirred for 1 more
hour (repeated once until full conversion was obtained). The
reaction was then evaporated to dryness in vacuo to afford 20.5 g
(97%) of the acid chloride 8c as a white solid; m/z (methyl ester
formed by addition of methanol prior to analysis)=516
(M+H).sup.+.
Step 3
##STR00085##
[0218] To a solution of (S)-4-benzyl-2-oxazolidinone (7.50 g, 42.3
mmol) in tetrahydrofuran (dry) (60 ml) under nitrogen atmosphere
n-butyllithium (26.4 ml, 42.3 mmol) was added slowly at -78.degree.
C. The reaction mixture was stirred for 40 minutes at -78.degree.
C. After 40 minutes, the anion solution was added via a canula to a
solution of the acid chloride 8c (20 g, 38.5 mmol) in 60 mL THF at
-78.degree. C. The reaction mixture was stirred for 1.5 hours at
-78.degree. C. When the reaction was finished, it was quenched with
an ammonia chloride solution at -70.degree. C. The reaction mixture
was then warmed up to room temperature and extracted with EtOAc,
washed with brine and dried over Na.sub.2SO.sub.4. The organic
layer was filtered and concentrated to afford 26.34 g of a yellow
solid. The two enantiomers 8d and 8e were separated by flash column
chromatography using 5:1 Heptane/EtOAc and were obtained as light
yellow solids; m/z=661 (M+H).sup.+.
Step 4
##STR00086##
[0220] Diastereoisomer 8d (11.17 g, 16.90 mmol) was first dissolved
in THF (130 ml) then methanol (130 ml) was added. 1N NaOH solution
(101 mL, 101 mmol) was added slowly so that the temperature was
kept below 30.degree. C. The reaction mixture was stirred at room
temperature for 2 hours. When the reaction was finished 1 N HCl
solution was added until the pH reached 2. 500 mL H.sub.2O was then
added and the reaction mixture was extracted with EtOAc, washed
with brine and concentrated. Purification by flash column
chromatography using 1:1 Heptane/EtOAc afforded 5.24 g (60%) of the
desired enantiomer
(4bR,5aS)-9-(tert-butoxycarbonyl)-12-cyclohexyl-3-methoxy-4b,5,5a,6-tetra-
hydrobenzo[3,4]cyclopropa[5,6]azepino[1,2-a]indole-5a-carboxylic
acid 8f with an ee of 97%; m/z=502 (M+H).sup.+.
Step 5
##STR00087##
[0222] To a stirred solution of intermediate 8f (2 g, 3.99 mmol) in
dry DMF (50 mL) at 0.degree. C., were added di-isopropyl ethylamine
(DIEA, 1.54 g, 11.9 mmol), HATU (2.27 g, 5.98 mmol) and
2,2'-oxybis(N-methylethanamine) (2.1 g, 15.95 mmol). The resulting
mixture was stirred at 0.degree. C. for 1 hour then kept at room
temperature for 12 hours. The reaction mixture was then
successively poured into an iced water solution, extracted with
dichloromethane, dried over MgSO.sub.4 then concentrated. The
residue was purified by column chromatography using a gradient of
methanol in dichloromethane (0 to 10%) to yield 0.94 g (38% yield)
of the desired product
tert-butyl(1aR,12bS)-8-cyclohexyl-11-methoxy-1a-(methyl{2-[2-(methylamino-
)ethoxy]ethyl}carbamoyl)-1,1a,2,12b-tetrahydrocyclopropa[d]indolo[2,1-a][2-
]benzazepine-5-carboxylate 8g as a white solid; m/z 616
(M+H).sup.+.
Step 6
##STR00088##
[0224] The synthesis of
(1aR,12bS)-8-cyclohexyl-11-methoxy-1a-(methyl{2-[2-(methyl-amino)ethoxy]e-
thyl}carbamoyl)-1,1a,2,12b-tetrahydrocyclopropa[d]indolo[2,1-a][2]-benzaze-
pine-5-carboxylic acid 8h was performed following the procedure
reported for the preparation of compound 1e, using intermediate 8g
instead of 1d. The obtained residue was further dissolved in DCM,
washed with water, dried over magnesium sulfate, filtered and
concentrated to dryness, leading to 0.474 g (56% yield) of title
compound 8h; m/z=560 (M+H).sup.+.
Step 7
##STR00089##
[0226] To a solution of intermediate 8h (0.474 g, 0.847 mmol) in
dioxane (10 mL) was added sulfamide (0.814 g, 8.47 mmol). The
resulting mixture was stirred at 100.degree. C. in a microwave oven
for 4 hours. The reaction mixture was then cooled down to room
temperature and concentrated. The residue was purified by column
chromatography using a gradient of methanol in dichloromethane (0
to 10%) to give 143 mg (26%) of the title product
(1aR,12bS)-1a-[(2-{2-[(aminosulfonyl)(methyl)amino]ethoxy}ethyl)
(methyl)carbamoyl]-8-cyclohexyl-11-methoxy-1,1a,2,12b-tetrahydrocycloprop-
a[d]indolo[2,1-a][2]benzazepine-5-carboxylic acid 8i; m/z=639
(M+H).sup.+.
Step 8
##STR00090##
[0228] The synthesis of
(1aR,12bS)-8-cyclohexyl-11-methoxy-16,22-dimethyl-1,12b-dihydro-5,1a-(met-
hanoiminothioiminoethanooxyethanoiminomethano)cyclopropa[d]-indolo[2,1-a][-
2]benzazepine-13,23(2H)-dione 15,15-dioxide 8 was performed
following the 2-step procedure reported for the synthesis of
compound 1, using intermediate 8i instead of 1e, yielding to 90 mg
(52% yield) of a white solid; m/z=621 (M+H).sup.+. .sup.1H NMR (400
MHz, CHLOROFORM-d) .delta. ppm 1.3-1.5 (m, 3H) 1.75-1.8 (m, 5H)
1.85-2.05 (m, 6H) 2.5-3 (m, 3H) 3.2 (s, 3H) 3.22 (s, 3H) 3.4-3.7
(m, 6H) 3.87 (s, 3H) 3.75-3.9 (m, 1H) 4.9-5.1 (m, 1H) 6.95-7.16 (d,
J=8.39 Hz, 1H) 7.28 (s, 1H) 7.44-7.55 (m, 2H) 7.80 (d, J=8.39 Hz,
1H) 9.4 (br. s., 1H).
Example 9
Synthesis of Compound 9
##STR00091##
[0229] Step 1
##STR00092##
[0231] Enantiomer
(4bS,5aR)-9-(tert-butoxycarbonyl)-12-cyclohexyl-3-methoxy-4b,5,5a,6-tetra-
hydrobenzo[3,4]cyclopropa[5,6]azepino[1,2-a]indole-5a-carboxylic
acid 9a was obtained in 27% yield, and 96% ee, following the
procedure reported for the synthesis of compound 8f, starting from
the diastereoisomer 8e instead of 8d; m/z=502 (M+H).sup.+.
Step 2
##STR00093##
[0233]
tert-butyl(1aS,12bR)-8-cyclohexyl-11-methoxy-1a-(methyl{2-[2-(methy-
lamino)ethoxy]ethyl}carbamoyl)-1,1a,2,12b-tetrahydrocyclopropa[d]indolo[2,-
1-a][2]benzazepine-5-carboxylate 9b was prepared in 60% yield from
9a and 2,2'-oxybis(N-methyl-ethanamine) following the procedure
used for the preparation of compound 1c; m/z=616 (M+H).sup.+.
Step 3
##STR00094##
[0235] To a solution of intermediate 9b (0.73 g, 1.185 mmol) in
dioxane (10 mL) was added sulfamide (1.14 g, 11.85 mmol). The
resulting mixture was stirred at 100.degree. C. in a microwave oven
for 3 hours. The reaction mixture was cooled down to room
temperature then concentrated. The residue was purified by column
chromatography using a gradient of methanol in dichloromethane (0
to 10%) to yield 743 mg (80%) of the title product
tert-butyl(1aS,12bR)-1a-[(2-{2-[(aminosulfonyl)(methyl)amino]-eth-
oxy}ethyl)(methyl)carbamoyl]-8-cyclohexyl-11-methoxy-1,1a,2,12b-tetrahydro-
-cyclopropa[d]indolo[2,1-a][2]benzazepine-5-carboxylate 9c.
Step 4
##STR00095##
[0237] The synthesis of
(1aS,12bR)-1a-[(2-{2-[(aminosulfonyl)(methyl)amino]ethoxy}ethyl)(methyl)c-
arbamoyl]-8-cyclohexyl-11-methoxy-1,1a,2,12b-tetrahydrocyclopropa[d]indolo-
[2,1-a][2]benzazepine-5-carboxylic acid 9d was performed following
the procedure reported for the preparation of compound 1e, using
intermediate 9c instead of 1d, yielding 517 mg (79% yield) of a
brownish foam; m/z=639 (M+H).sup.+.
Step 5
##STR00096##
[0239] The synthesis of (1
aS,12bR)-8-cyclohexyl-11-methoxy-16,22-dimethyl-1,12b-dihydro-5,1a-(metha-
noiminothioiminoethanooxyethanoiminomethano)cyclopropa[d]indolo-[2,1-a][2]-
benzazepine-13,23(2H)-dione 15,15-dioxide 9 was performed following
the 2-step procedure reported for the synthesis of compound 1,
using intermediate 9d instead of 1e, yielding to 80 mg (16% yield)
of a white solid; m/z=621 (M+H).sup.+. .sup.1H NMR (400 MHz,
CHLOROFORM-d) .delta. ppm 1.3-1.5 (m, 3H) 1.75-1.8 (m, 5H)
1.85-2.05 (m, 6H) 2.5-3 (m, 3H) 3.2 (s, 3H) 3.22 (s, 3H) 3.4-3.7
(m, 6H) 3.87 (s, 3H) 3.75-3.9 (m, 1H) 4.9-5.1 (m, 1H) 6.95-7.16 (d,
J=8.39 Hz, 1H) 7.28 (s, 1H) 7.44-7.55 (m, 2H) 7.80 (d, J=8.39 Hz,
1H) 9.4 (br. s., 1H).
Example 10
Synthesis of Compound 10
##STR00097##
[0240] Step 1
##STR00098##
[0242] To a solution of 5-tert-butyl 1a-methyl
8-cyclohexyl-11-methoxy-1,12b-dihydrocyclopropa[d]indolo[2,1-a][2]benzaze-
pine-1a,5(2H)-dicarboxylate 8a (2 g, 3.88 mmol) in dichloromethane
(25 mL) was added TFA (22.34 g, 194 mmol). The resulting mixture
was stirred at room temperature for 6 hours then concentrated to
dryness. The residue was successively dissolved in dichloromethane,
washed with water, dried under MgSO.sub.4, filtered and
concentrated. The residue was then purified by column
chromatography using dichloromethane and ethyl acetate as eluent to
yield 1.7 g (95%) of the title product
8-cyclohexyl-11-methoxy-1a-(methoxycarbonyl)-1,1a,2,12b-tetrahydrocyclopr-
opa[d]indolo[2,1-a][2]benzazepine-5-carboxylic acid 10a as a white
powder; m/z 460 (M+H).sup.+.
Step 2
##STR00099##
[0244] 1,1'-Carbonyldiimidazole (0.847 g, 5.22 mmol) was added to a
stirred solution of
8-cyclohexyl-11-methoxy-1a-(methoxycarbonyl)-1,1a,2,12b-tetrahydrocyclopr-
opa[d]-indolo[2,1-a][2]benzazepine-5-carboxylic acid 10a (0.8 g,
1.74 mmol) in THF (15 mL) at 25.degree. C. The evolution of
CO.sub.2 was instantaneous and when it slowed the solution was
heated at 50.degree. C. for 2 hours and then cooled to room
temperature.
Tert-butyl{4-[(amino-sulfonyl)(methyl)amino]butyl}carbamate 10b
(0.735 g, 2.61 mmol) was added followed by the addition of DBU
(0.53 g, 3.48 mmol). Stirring was continued for 12 hours at
50.degree. C. The mixture was cooled to room temperature then
partitioned between dichloromethane and water. The water was
extracted with dichloromethane. The organic layers were dried over
MgSO.sub.4 then concentrated to dryness. The residue was purified
by column chromatography using dichloromethane and ethyl acetate to
yield 0.66 g (53%) of the title product methyl
5-({[{4-[(tert-butoxycarbonyl)amino]butyl}(methyl)amino]sulfonyl}carbamoy-
l)-8-cyclohexyl-11-methoxy-1,12b-dihydrocyclopropa[d]indolo[2,1-a][2]benza-
zepine-1a(2H)-carboxylate 10c as a white foam; m/z 723
(M+H).sup.+
Step 3
##STR00100##
[0246] To a solution of intermediate 10c (0.65 g, 0.899 mmol) in
THF (20 mL) was added lithium hydroxide (0.75 g, 1.8 mmol)
dissolved in water (5 mL). The resulting mixture was stirred at
room temperature overnight then diluted with water and neutralized
with a 2M HCl aqueous solution. The resulting mixture was extracted
with dichloromethane, dried over MgSO.sub.4 then concentrated. The
residue was purified by column chromatography using a gradient of
methanol in CH.sub.2Cl.sub.2 to yield 0.55 g (86%) of the title
product
5-({[{4-[(tert-butoxycarbonyl)amino]butyl}(methyl)amino]sulfonyl}-carbamo-
yl)-8-cyclohexyl-11-methoxy-1,12b-dihydrocyclopropa[d]indolo[2,1-a][2]benz-
azepine-1a(2H)-carboxylic acid 10d as a white solid; m/z 709
(M+H).sup.+
Step 4
##STR00101##
[0248] TFA (2.5 g, 22 mmol) was added to a solution of intermediate
10d (0.52 g, 0.734 mmol) in DCM (10 mL). The resulting mixture was
stirred at RT for approximately 10 hours. The reaction was then
evaporated to dryness and the residue was purified by column
chromatography using a gradient of methanol in DCM to afford 0.3 g
(68%) of the title compound
5-({[(4-aminobutyl)(methyl)amino]sulfonyl}carbamoyl)-8-cyclohexyl-11-meth-
oxy-1,12b-dihydrocyclopropa[d]indolo[2,1-a][2]benzazepine-1a(2H)-carboxyli-
c acid 10e as a TFA salt; m/z 609 (M+H).sup.+
Step 5
##STR00102##
[0250] To a stirred solution of intermediate 10e (0.22 g, 0.36
mmol) in dry DMF (100 mL), at 0.degree. C., were added DIPEA (0.14
g, 1.08 mmol) and HATU (0.206 g, 0.542 mmol). The resulting mixture
was stirred at 0.degree. C. for 1 hour then kept at room
temperature for 12 hours. The reaction mixture was then
successively poured into an iced watered solution, extracted with
dichloromethane, dried over MgSO.sub.4 and then concentrated. The
residue was purified by column chromatography to yield 0.188 g
(88%) of the title product
8-cyclohexyl-11-methoxy-16-methyl-1,12b-dihydro-5,1a-(methanoiminothioimi-
nobutanoimino)cyclopropa[d]indolo[2,1-a][2]benzazepine-13,22(2H)-dione
15,15-dioxide 10 as a white solid. .sup.1H NMR (DMSO-d.sub.6): 11.5
(s, 1H), 8.4 (s, 1H), 8.3 (s, 1H), 7.8 (d, J=8.2 Hz, 1H), 7.3 (d,
J=8.2 Hz, 1H), 7.25 (d, J=8.4 Hz, 1H), 7.15 (s, 1H), 7 (d, J=8.4
Hz, 1H), 5.6 (d, J=16 Hz, 1H), 3.85 (s, 3H), 3.55 (d, J=16 Hz, 1H),
3-3.2 (m, 2H), 3 (s, 3H), 2.7-2.9 (m, 4H), 1.8-2.1 (m, 5H), 1.6-1.7
(m, 2H), 1.3-1.6 (m, 6H), 1-0.7 (m, 3H); m/z 609 (M+H).sup.+
Example 11
Synthesis of Compound 11
##STR00103##
[0251] Step 1
##STR00104##
[0253] 1,1'-carbonyldiimidazole (0.522 g, 3.22 mmol) was added to a
stirred solution of
8-cyclohexyl-11-methoxy-1a-(methoxycarbonyl)-1,1a,2,12b-tetrahydrocyclopr-
opa-[d]indolo[2,1-a][2]benzazepine-5-carboxylic acid 10a (0.74 g,
1.61 mmol) in THF (15 mL) at 25.degree. C. The evolution of
CO.sub.2 was instantaneous and when it slowed the solution was
heated at 50.degree. C. for 2 hours and then cooled to room
temperature. Tert-butyl
4-{2-[(aminosulfonyl)(methyl)amino]ethyl}piperazine-1-carboxylate
11a (1.038 g, 3.22 mmol) was added followed by the addition of DBU
(0.49 g, 3.22 mmol). Stirring was continued for 12 hours at
50.degree. C. The mixture was cooled to room temperature then
partitioned between dichloromethane and water. The water was
extracted with dichloromethane and the organic layers were dried
over MgSO.sub.4 then concentrated to dryness. The residue was
purified by column chromatography using dichloromethane and ethyl
acetate to yield 0.83 g (68%) of the title compound methyl
5-({[{2-[4-(tert-butoxycarbonyl)piperazin-1-yl]ethyl}(methyl)amino]sulfon-
yl}carbamoyl)-8-cyclohexyl-11-methoxy-1,12b-dihydrocyclopropa[d]indolo[2,1-
-a][2]benzazepine-1a(2H)-carboxylate 11b as a white foam; m/z 764
(M+H).sup.+
Step 2
##STR00105##
[0255] To a solution of intermediate 11b (0.6 g, 0.785 mmol) in THF
(20 mL) was added LiOH (0.82 g, 1.96 mmol) in water (5 mL). The
resulting mixture was stirred at room temperature overnight then
diluted with water and neutralized with a 2M HCl aqueous solution.
The resulting mixture was extracted with dichloromethane, dried
over MgSO.sub.4 then concentrated. The resulting residue was
purified by column chromatography using CH.sub.2Cl.sub.2 and
methanol to yield 0.4 g (68%) of the title compound
5-({[{2-[4-(tert-butoxycarbonyl)piperazin-1-yl]ethyl}(methyl)amino]sulfon-
yl}carbamoyl)-8-cyclohexyl-11-methoxy-1,12b-dihydrocyclopropa[d]indolo[2,1-
-a][2]benzazepine-1a(2H)-carboxylic acid 11c as a white solid; m/z
750 (M+H).sup.+
Step 3
##STR00106##
[0257] TFA (1.44 g, 12.7 mmol) was added to a solution of
intermediate 11c (0.38 g, 0.507 mmol) in dichloromethane (10 mL).
The resulting mixture was stirred at RT for approximately 10 hours.
The reaction was then evaporated to dryness and the residue was
purified by column chromatography using dichloromethane and
methanol to afford the title compound
8-cyclohexyl-11-methoxy-5-({[methyl(2-piperazin-1-yl-ethyl)amino-
]sulfonyl}carbamoyl)-1,12b-dihydrocyclopropa[d]indolo[2,1-a][2]-benzazepin-
e-1a(2H)-carboxylic acid 11d (0.24 g, 73%); m/z 650 (M+H).sup.+
Step 4
##STR00107##
[0259] To a stirred solution of intermediate 11d (0.24 g, 0.37
mmol) in dry DMF (100 mL), at 0.degree. C., were added di-isopropyl
ethylamine (0.143 g, 1.1 mmol) and HATU (0.211 g, 0.554 mmol). The
resulting mixture was stirred at 0.degree. C. for 1 hour then kept
at room temperature for 12 hours. The reaction mixture was then
successively poured into an iced watered solution, extracted with
dichloromethane, dried over MgSO.sub.4 and concentrated. The
residue was purified by column chromatography using
dichloro-methane/methanol to yield 0.018 g (18%) of the title
compound
31-cyclohexyl-8-methoxy-22-methyl-21-thia-1,13,20,22,25-pentaazaheptacycl-
o-[23.2.2.1.sup.3,13.1.sup.12,15.1.sup.14,18.0.sup.3,5.0.sup.6,11]dotriaco-
nta-6,8,10,12(31),14(30),15,17-heptaene-2,19-dione 21,21-dioxide 11
as a white solid; m/z 632 (M+H).sup.+
Example 12
Synthesis of Compound 12
##STR00108##
[0260] Step 1
##STR00109##
[0262] To a solution of
5-tert-butyl-1a-methyl-8-cyclohexyl-11-methoxy-1,12b-dihydrocyclopropa[d]-
indolo[2,1-a][2]benzazepine-1a,5(2H)-dicarboxylate 8a (2 g, 3.88
mmol) in dichloromethane (25 mL) was added trifluoroacetic acid
(22.34 g, 194 mmol). The resulting mixture was stirred at room
temperature for 6 hours then concentrated to dryness. The residue
was successively dissolved in dichloromethane, washed with water,
dried under MgSO.sub.4, filtered and concentrated. The residue was
purified by column chromatography using dichloromethane and ethyl
acetate as eluent to yield 1.7 g (95%) of the title product
8-cyclohexyl-11-methoxy-1a-(methoxycarbonyl)-1,1a,2,12b-tetrahydrocyclopr-
opa[d]indolo[2,1-a][2]benzazepine-5-carboxylic acid 12a as a white
powder; m/z 460 (M+H).sup.+
Step 2
##STR00110##
[0264] To a solution of intermediate 12a (1.73 g, 3.76 mmol) in THF
(25 mL) at 0.degree. C. were added successively
4-dimethylaminopyridine (DMAP) (1.38 g, 3.76 mmol),
N1-((ethylimino)methylene)-N3,N3-dimethylpropane-1,3-diamine
hydrochloride (EDC) (2.16 g, 11.29 mmol) and
allyl(methyl)aminosulfonamide 12b (1.3 g, 8.66 mmol). The resulting
mixture was stirred at 0.degree. C. for 2 h then at room
temperature for 8 h. Water was then added and the reaction mixture
was filtered. The resulting solid was purified by column
chromatography using dichloromethane and ethyl acetate to yield 500
mg (23%) of the title product methyl
5-({[allyl(methyl)amino]sulfonyl}-carbamoyl)-8-cyclohexyl-11-methoxy-1,12-
b-dihydrocyclopropa[d]indolo[2,1-a][2]-benzazepine-1a(2H)-carboxylate
12c; m/z 592 (M+H).sup.+
Step 3
##STR00111##
[0266] To a solution of intermediate 12c (0.5 g, 0.845 mmol) in THF
(20 mL) was added lithium hydroxide (0.73 g, 1.69 mmol) in water (5
mL). The resulting mixture was stirred at room temperature
overnight then diluted with water and neutralized with a 2M HCl
aqueous solution. The resulting mixture was extracted with
dichloromethane, dried over MgSO.sub.4 then concentrated. The
resulting residue was purified by column chromatography using
CH.sub.2Cl.sub.2 and methanol to yield 0.4 g (75%) of the title
product
5-({[allyl(methyl)amino]sulfonyl}carbamoyl)-8-cyclohexyl-11-metho-
xy-1,12b-dihydrocyclopropa[d]indolo[2,1-a][2]benzazepine-1a(2H)-carboxylic
acid 12d as a white solid; m/z 578 (M+H).sup.+
Step 4
##STR00112##
[0268] To a solution of intermediate 12d (0.2 g, 0.346 mmol) in THF
(15 mL), at 0.degree. C., were added successively
4-dimethylaminopyridine (DMAP) (0.127 g, 1.04 mmol),
N1-((ethylimino)methylene)-N3,N3-dimethylpropane-1,3-diamine
hydrochloride (EDC) (0.199 g, 1.04 mmol) and but-3-en-1-amine
(0.062 g, 0.866 mmol). The resulting mixture was stirred at
0.degree. C. for 2 h then at room temperature for 8 h. Water was
then added and the resulting mixture was filtered. The solid was
washed with dichloromethane then the filtrate was successively
extracted with dichloromethane, dried over MgSO.sub.4, filtered and
concentrated. The resulting residue was purified by column
chromatography with dichloromethane and ethyl acetate to yield 70
mg (32%) of the title product
N.sup.5-{[allyl(methyl)amino]sulfonyl}-N.sup.1a-but-3-en-1-yl-8-cyclohexy-
l-11-methoxy-1,12b-dihydrocyclopropa[d]indolo[2,1-a][2]benzazepine-1a,5(2H-
)-dicarboxamide 12e; m/z 631 (M+H).sup.+
Step 5
##STR00113##
[0270] A solution of intermediate 12e (0.1 g, 0.16 mmol) in
dichloroethane (50 mL) was degassed with argon for 10 minutes then
Hoveyda-Grubbs 1.sup.st generation catalyst (0.03 mg, 0.032 mmol)
was added. The resulting mixture was warmed to 70.degree. C. and
kept under argon overnight. The mixture was then cooled down to
room temperature and the solvent was removed under vacuum. The
resulting dark residue was purified by column chromatography using
DCM and ethyl acetate to give 15 mg (16%) of the title product
8-cyclohexyl-11-methoxy-16-methyl-1,12b-dihydro-5,1a-(methaniminothio-imi-
nopent[2]enoiminomethano)cyclopropa[d]indolo[2,1-a][2]benzazepine-13,23(2H-
)-dione 15,15-dioxide 12 as a white solid; m/z 603 (M+H).sup.+
Example 13
Synthesis of Compound 13
##STR00114##
[0271] Step 1
##STR00115##
[0273]
13-Cyclohexyl-3-methoxy-7H-benzo[3,4]azepino[1,2-a]indole-6,10-dica-
rboxylic acid 10-tert-butyl ester 6-methyl ester 1a (1 g, 1 eq) was
dissolved in dry dichloromethane under N.sub.2, followed by the
addition of trifluoroacetic acid (TFA) (8.88 ml, 60 eq). The
solution was stirred at RT for 24 h. The solvent was then removed
under reduced pressure. The crude product was triturated with
diethyl ether. The crystals were filtered off and dried under
vacuum overnight to afford the title product
13-Cyclohexyl-3-methoxy-7H-benzo[3,4]azepino[1,2-a]indole-6,10-dicarboxyl-
ic acid 6-methyl ester 13a (89%, 0.86 g); LC-MS: Rt. 3.19 min., m/z
446 [M+H].sup.+.
Step 2
##STR00116##
[0275]
13-Cyclohexyl-3-hydroxy-7H-benzo[3,4]azepino[1,2-a]indole-6,10-dica-
rboxylic acid 6-methyl ester 13a (0.86 g, 1 eq),
N-methyl-N-allyl-sulfuric diamide 12b (0.67 g, 2.03 eq),
N.sup.1-((ethylimino)methylene)-N.sup.3,N3-dimethylpropane-1,3-diamine
hydrochloride (EDCI) (1.14 g, 3.06 eq) and
dimethyl-pyridin-4-yl-amine (DMAP) (0.67 g, 3.04 eq) were dissolved
in dry dimethylformamide (20 ml) under N.sub.2. The solution was
stirred at RT for 3 days. This solution was slowly added into ice
water. The water layer was extracted with ethyl acetate (3.times.50
ml) and washed with tetrahydrofurane (3.times.50 ml). The combined
organic layers were dried over magnesium sulfate, filtered and
evaporated under reduced pressure. The crude product was purified
by preparative HPLC to give 0.63 g (55%) of the title product 13b;
LC-MS: Rt. 6.16 min., m/z 578 [M+H].sup.+. .sup.1H-NMR (DMSO)
.delta. (ppm) 1.13-1.20 (m, 1H, CH.sub.2), 1.30-1.47 (m, 3H,
CH.sub.2(2x)), 1.62-1.78 (m, 2H, CH.sub.2), 1.81-1.93 (m, 1H,
CH.sub.2), 1.93-2.12 (m, 3H, CH.sub.2(2x)), 2.70-2.82 (m, 1H, CH),
2.86 (s, 3H, CH.sub.3N), 3.79 (s, 3H, CH.sub.3O), 3.88 (s, 3H,
CH.sub.3O), 3.90-3.98 (m, 2H, CH.sub.2), 4.21 (d, 1H, J=12.97 Hz,
CH.sub.2), 5.21 (d, 1H, J=10.15 Hz, CH.sub.2), 5.31 (d, 1H, J=17.13
Hz, CH.sub.2), 5.61 (d, 1H, J=13.12 Hz, CH.sub.2), 5.78-5.90 (m,
1H, CH.sub.arom), 7.25 (dd, 1H, J=2.50 and J=8.60 Hz, CH.sub.arom),
7.32-7.35 (m, 1H, CH.sub.arom), 7.54 (d, 1H, J=8.60 Hz,
CH.sub.arom), 7.61 (d, 1H, J=8.45 Hz, CH.sub.arom), 7.88 (d, 1H,
J=9.01 Hz, CH.sub.arom), 7.91 (s, 1H, CH), 8.31-8.34 (brs, 1H,
NHSO.sub.2).
Step 3
##STR00117##
[0277] Compound 13b (0.60 g, 1 eq) was dissolved in a mixture of
tetrahydrofurane:methanol (1:1) (20 ml), followed by the addition
of a LiOH solution in water (0.09 g, 2 eq). The solution was
stirred overnight at RT for several days. The solvents were then
evaporated under reduced pressure and the water layer was acidified
with a 3 N HCl solution until pH 2. The resulting crystals were
filtered off, washed with water and isopropyl ether and dried under
vacuum overnight to afford 0.44 g (74%) of the title product 13c;
LC-MS: Rt. 5.84 min., m/z 562 [M-H].sup.-.
Step 4
##STR00118##
[0279] Compound 13c (0.44 g, 1 eq) and HATU (0.47 g, 1.6 eq) were
dissolved in dimethylformamide under N.sub.2, followed by the
addition of DIPEA (0.15 g, 0.20 ml, 1.5 eq) and allylamine (0.07
ml, 1.2 eq). The solution was stirred at RT for 3 days. The
dimethylformamide solution was then slowly poured into ice water.
The resulting crystals were filtered off, washed with water and
dried under vacuum overnight to afford 0.47 g (100%) of the title
product 13d; LC-MS: Rt. 3.01 min., m/z 603 [M+H].sup.+.
Step 5
##STR00119##
[0281] N.sub.2 was bubbled through a solution of compound 13d (0.47
g, 1 eq) in 50 ml of dichloroethane for 1 h. Then Grubbs 2.sup.nd
generation catalyst (0.13 g, 0.2 eq) was added and the reaction
mixture was heated at 80.degree. C. overnight. The solution was
cooled down to RT and some extra amount of catalyst was added (65
mg). The solution was heated at 80.degree. C. under N.sub.2 for
several hours. The solution was then evaporated under reduced
pressure. The product was purified by flash chromatography on
elution of dichloromethane:methanol (100 to 95:5), and was
subsequently recrystallized from methanol. Finally the product was
purified by preparative HPLC chromatography to afford 30 mg (5.86%)
of the title product 13; LC-MS: Rt. 5.33 min., m/z 575 [M+H].sup.+.
.sup.1H-NMR (DMSO) .delta. (ppm) 1.05-1.21 (m, 1H, CH.sub.2),
1.30-1.48 (m, 3H, CH.sub.2 (2x)), 1.62-1.78 (m, 2H, CH.sub.2),
1.82-1.93 (m, 1H, CH.sub.2), 1.93-2.12 (m, 3H, CH.sub.2 (2x)),
2.65-2.90 (m, 4H, CH and CH.sub.3N), 3.56 (d, 2H, J=18.10 Hz,
CH.sub.2), 3.80-3.97 (brs, 5H, CH.sub.2 and CH.sub.3O), 4.21 (d,
1H, J=15.12 Hz, CH.sub.2), 4.28-4.46 (m, 1H, CH), 5.72 (d, 1H,
J=14.15 Hz, CH.sub.2), 5.78-5.88 (m, 1H, CH), 6.53 (s, 1H,
CH.sub.arom), 7.18-7.28 (m, 2H, CH.sub.arom (2x)), 7.39-7.49 (m,
1H, CH.sub.arom), 7.55 (d, 1H, J=8.38 Hz, CH.sub.arom), 7.62 (s,
1H, CH.sub.arom), 7.72-7.84 (m, 1H, NH), 8.29 (s, 1H, CH),
8.51-8.62 (brs, 1H, NH).
Example 14
Synthesis of Compound 14
##STR00120##
[0282] Step 1
##STR00121##
[0284]
13-Cyclohexyl-3-methoxy-7H-benzo[3,4]azepino[1,2-a]indole-6,10-dica-
rboxylic acid 6-methyl ester 13a (0.60 g, 1 eq) was dissolved in
dry acetonitrile (50 ml) under N.sub.2, followed by the addition of
di-imidazol-1-yl-methanone (CDI) (0.66 g, 3 eq). The solution was
stirred overnight at 50.degree. C. The solvent was then evaporated
under reduced pressure and the crude product was purified by flash
chromatography on elution with heptane:acetonitrile and finally
ethyl acetate. The product was recrystallized from ethyl acetate to
give 0.50 g (75%) of the title product 14a.
Step 2
##STR00122##
[0286] Compound 14a (0.50 g, 1 eq) was dissolved in dry
acetonitrile (50 ml), followed by the addition of tert-butyl
4-(2-(sulfamoylamino)ethyl)piperazine-1-carboxylate 14b (0.47 g,
1.50 eq) and 2,3,4,6,7,8,9,10-octahydro-pyrimido[1,2-a]azepine
(DBU) (0.31 g, 2 eq). The solution was heated at 50.degree. C.
overnight, then evaporated under reduced pressure. The resulting
residue was stirred in a 0.1 N citric acid water solution. The
crystals were filtered off and dried under vacuum overnight. The
product was purified by column chromatography on elution with
dichloromethane to remove the first impurity. The other obtained
fractions were added together. This product was further purified by
flash chromatography on elution with dichloromethane:methanol (100
to 99:1) to oafford 0.41 g (55%) of the title product 14c; LC-MS:
Rt. 5.59 min., m/z 736 [M+H].sup.+. .sup.1H-NMR (CDCl.sub.3)
.delta. (ppm) 1.18-1.34 (m, 1H, CH.sub.2), 1.35-1.50 (brs, 10H,
CH.sub.2 and C(CH.sub.3).sub.3), 1.70-1.85 (m, 3H, CH.sub.2(2x)),
1.90-2.12 (m, 5H, CH.sub.2 (3x)), 2.30-2.41 (m, 4H, CH.sub.2 (2x)),
2.52-2.62 (m, 2H, CH.sub.2), 2.77-2.90 (m, 1H, CH), 3.13-3.22 (m,
2H, CH.sub.2), 3.43-3.57 (m, 4H, CH.sub.2 (2x)), 3.83 (s, 3H,
CH.sub.3O), 3.92 (s, 3H, CH.sub.3O), 4.16-4.23 (m, 1H, CH.sub.2),
5.58-5.69 (m, 1H, CH.sub.2), 7.00 (d, 1H, J=2.54 Hz, CH.sub.arom),
7.11 (dd, 1H, J=2.67 and J=8.59 Hz, CH.sub.arom), 7.48 (d, 1H,
J=8.44 Hz, CH.sub.arom), 7.53 (d, 1H, J=8.61 Hz, CH.sub.arom), 7.83
(s, 1H, CH.sub.arom), 7.90 (d, 1H, J=8.48 Hz, CH.sub.arom), 8.09
(s, 1H, CH).
Step 3
##STR00123##
[0288] Compound 14c (0.41 g, 1 eq) was dissolved in dry
dichloromethane (10 ml) under N.sub.2 followed by the addition of
trifluoroacetic acid (1.30 ml, 30 eq). The solution was stirred at
RT overnight. The solvent was then removed under reduced pressure
and the crude product was stirred in diethyl ether. The resulting
crystals were filtered off and dried under reduced pressure to
afford 0.31 g (87%) of the title product 14d; LC-MS: RT. 3.81 min.,
m/z 634 [M-H].sup.-.
Step 4
##STR00124##
[0290] Compound 14d (0.31 g, 1 eq) was dissolved in a mixture of
tetrahydrofurane:methanol (1:1), followed by the addition of 50%
NaOH-water solution (1 ml). The solution was stirred at RT
overnight then evaporated under reduced pressure. The water layer
was acidified with acetic acid to pH 4, and extracted with ethyl
acetate (7.times.50 ml). The combined ethyl acetate layers were
dried over sodium sulfate, filtered off and evaporated under
reduced pressure to obtain the desired compound 14e as a yellow
powder (0.30 g, 100%); LC-MS: Rt. 3.64 min., m/z 622
[M+H].sup.+.
Step 5
##STR00125##
[0292] The synthesis of the title compound 14 is being performed
following the procedure reported for the synthesis of compound 11,
using intermediate 14e instead of 11d.
Example 15
Synthesis of Compound 15
##STR00126##
[0293] Step 1
##STR00127##
[0295] Compound 13a (0.20 g, 1 eq) was dissolved in dry
acetonitrile under N.sub.2, followed by the addition of CDI (0.1 g,
1.3 eq). The solution was stirred at 60.degree. C. for 1 h.
According to TLC, the reaction went to completion. DBU (0.10 ml,
1.52 eq) and diaminosulfuric diamide 15a (0.29 g, 2 eq) were then
added. The solution was stirred at 60.degree. C. for 3 h, then was
evaporated under reduced pressure. A citric acid water solution
(0.1 N) cooled in ice, was added to the crude product. The residual
solution was extracted with ethyl acetate (3.times.50 ml). The
combined organic layers were washed with brine (50 ml), dried over
sodium sulfate, filtered off and evaporated under reduced pressure
to afford 0.21 g (62%) of the title product 15b; LC-MS: Rt: 5.63
min., m/z 750 [M+H].sup.+.
Step 2
##STR00128##
[0297] The synthesis of compound 15c was performed following the
procedure reported for the synthesis of compound
5-({[{2-[4-(tert-butoxycarbonyl)piperazin-1-yl]ethyl}(methyl)amino]sulfon-
yl}carbamoyl)-8-cyclohexyl-11-methoxy-1,12b-dihydrocyclopropa[d]indolo[2,1-
-a][2]benzazepine-1a(2H)-carboxylic acid (11c), using intermediate
15b instead of methyl
5-({[{2-[4-(tert-butoxycarbonyl)piperazin-1-yl]ethyl}(methyl)amino]sulfon-
yl}carbamoyl)-8-cyclohexyl-11-methoxy-1,12b-dihydrocyclopropa[d]indolo[2,1-
-a][2]benzazepine-1a(2H)-carboxylate (11b); m/z 736
[M+H].sup.+.
Step 3
##STR00129##
[0299] The synthesis of the title compound 15d was performed
following the procedure reported for the synthesis of compound
8-cyclohexyl-11-methoxy-5-({[methyl-(2-piperazin-1-ylethyl)amino]sulfonyl-
}carbamoyl)-1,12b-dihydrocyclopropa[d]-indolo[2,1-a][2]benzazepine-1a(2H)--
carboxylic acid (11d), using intermediate 15c instead of
5-({[{2-[4-(tert-butoxycarbonyl)piperazin-1-yl]ethyl}(methyl)amino]-sulfo-
nyl}carbamoyl)-8-cyclohexyl-11-methoxy-1,12b-dihydrocyclopropa[d]indolo-[2-
,1-a][2]benzazepine-1a(2H)-carboxylic acid (11c), yielding to 448
mg (quantitative yield) of the desired product; m/z 636
[M+H].sup.+.
Step 4
##STR00130##
[0301] The synthesis of the title compound 15 was performed
following the procedure reported for the synthesis of compound
31-cyclohexyl-8-methoxy-22-methyl-21-thia-1,13,20,22,25-pentaazaheptacycl-
o[23.2.2.1.sup.3,13.1.sup.12,15.1.sup.14,18.0.sup.3,5.0.sup.6,11]dotriacon-
ta-6,8,10,12(31),14(30),15,17-heptaene-2,19-dione 21,21-dioxide 11,
using intermediate 15d instead of
8-cyclohexyl-11-methoxy-5-({[methyl(2-piperazin-1-ylethyl)amino]-sulfonyl-
}carbamoyl)-1,12b-dihydrocyclopropa[d]indolo[2,1-a][2]benzazepine-1a(2H)-c-
arboxylic acid 11d, yielding 150 mg (34% yield) of a cream solid;
m/z 618 [M+H].sup.+. .sup.1H NMR (400 MHz, DMSO-d6) .delta. ppm
1.06-1.18 (m, 1H) 1.19-1.31 (m, 2H) 1.31-1.50 (m, 2H) 1.62-1.78 (m,
2H) 1.81-1.93 (m, 1H) 1.93-2.10 (m, 2H) 2.53-3.21 (m, 12H)
3.31-3.67 (m, 4H) 3.86 (s, 3H) 4.33-4.51 (m, 1H) 4.99-5.16 (m, 1H)
7.06-7.14 (m, 2H) 7.17 (d, J=8.02 Hz, 1H) 7.52 (d, J=8.22 Hz, 1H)
7.55-7.68 (m, 1H) 7.77 (m, 1H) 8.39 (m, 1H).
Example 16
Synthesis of Compound 16
##STR00131##
[0302] Step 1
##STR00132##
[0304] A solution of 50% NaOH w/w in water (9.31 g) was added to a
stirred solution of 16a (3.0 g, 5.82 mmoles) in THF (100 mL) and
MeOH (150 mL). After 1 hour the reaction mixture was concentrated
under reduced pressure, and subsequently diluted with ice-cold
water (150 mL). The pH of the resulting solution was adjusted to 6
with diluted HCl. A precipitate was formed, which was collected by
filtration, washed with water and dried under vacuum to give 3.17 g
(89%) of 16b as a yellowish powder. The product was used without
any further purification in the next step; m/z=502 (M+H).sup.+.
Step 2
##STR00133##
[0306] HATU (3.6 g, 9.48 mmol) was added under nitrogen to a
stirred solution of 16b (3.17 g, 6.32 mmol), DIPEA (3.3 mL, 3 eq)
and 2,2'-oxybis(N-methylethanamine) (3.34 g, 4 eq) in 60 mL of dry
THF. After 1 h, the reaction mixture was quenched with water (100
mL) and extracted with ethyl acetate (EtOAc). The organic layer was
successively dried (Na.sub.2SO.sub.4), filtered and evaporated. The
residue was triturated in water, filtered and dried to give 4.05 g
(quantitative yield) of the target compound 16c, used directly in
the next step: m/z=616 (M+H).sup.+
Step 3
##STR00134##
[0308] A solution of 16c (3.90 g, 6.33 mmol) and sulfamide (3.04 g,
6 eq) in dioxane (100 mL) was refluxed at 100.degree. C. overnight.
The reaction mixture was cooled down to room temperature, then
evaporated under vacuum. The residue was redissolved in DCM, washed
with water, dried over magnesium sulfate, filtered and concentrated
to give 4.48 g (quantitative yield) of the desired product 16d,
used directly in the next step: m/z=695 (M+H).sup.+
Step 4
##STR00135##
[0310] TFA (14.7 g, 129 mmol) was added to a solution of 16d (4.48
g, 6.45 mmol) in dichloromethane (50 mL). After 1 h, the reaction
mixture was concentrated under vacuum. The residue was triturated
in ether, filtered and washed with ether, then purified by
chromatography (gradient EtOAc to EtOAc/EtOH, 9:1) to give 3.05 g
(68%) of the desired product 16e: m/z=639 (M+H).sup.+
Step 5
##STR00136##
[0312] Carbonyldiimidazole (1.07 g, 6.59 mmol) was added to a
stirred solution of 16e (3.05 mg, 4.39 mmol) in dry ACN (40 mL).
The reaction mixture was stirred at 60.degree. C. for 1 h: complete
conversion to the acyl imidazole intermediate was observed. The
resulting solution was cooled down to RT, diluted with dry ACN (300
mL) and DBU (1.34 g, 2 eq) was added. The reaction mixture was
stirred overnight at room temperature, then concentrated under
reduced pressure. The residue was redissolved in DCM, washed with
water, dried, filtered and concentrated. Purification by column
chromatography (gradient DCM to DCM/MeOH 9:1) provided 930 mg (33%)
of the title product 16 as a white powder: m/z=621 (M+H).sup.+,
.sup.1H NMR (400 MHz, CHLOROFORM-d) .delta. ppm 1.15-1.31 (m, 1H)
1.31-1.52 (m, 3H) 1.69-1.81 (m, 2H) 1.84 (s, 3H) 1.88-2.13 (m, 7H)
2.45 (d, J=14.87 Hz, 1H) 2.76-2.92 (m, 1H) 3.14 (s, 3H) 3.40 (d,
J=15.65 Hz, 1H) 3.54-3.70 (m, 3H) 3.81-3.90 (m, 1H) 3.93 (s, 3H)
4.03-4.18 (m, 1H) 4.37 (d, J=14.67 Hz, 1H) 4.64-4.80 (m, 2H) 7.06
(d, J=8.80 Hz, 1H) 7.09 (s, 1H) 7.48 (d, J=8.22 Hz, 1H) 7.57 (s,
1H) 7.70 (d, J=8.22 Hz, 1H) 7.89 (d, J=8.41 Hz, 1H) 10.01 (br. s.,
1H)
Example 17
Synthesis of Compound 17
##STR00137##
[0313] Step 1
##STR00138##
[0315] The synthesis of the title compound 17a was performed
following the procedure reported for the synthesis of compound 16c,
using N.sup.1,N.sup.4-dimethylbutane-1,4-diamine instead of
2,2'-oxybis(N-methylethanamine), yielding 1.25 g (quant. yield) of
a white solid; m/z 600 [M+H].sup.+.
Step 2
##STR00139##
[0317] The synthesis of the title compound 17b was performed
following the procedure reported for the synthesis of compound 16d,
using compound 17a instead of compound 16c, yielding 1 g (54%
yield) of a slightly yellow solid; m/z 679 [M+H].sup.+.
Step 3
##STR00140##
[0319] The synthesis of the title compound 17c was performed
following the procedure reported for the synthesis of compound 16e,
using compound 17b instead of compound 16d, yielding 538 mg (62%
yield) of a slightly brown solid; m/z 623 [M+H].sup.+.
Step 4
##STR00141##
[0321] The synthesis of the title compound 17 was performed
following the procedure reported for the synthesis of compound 16,
using compound 17c instead of compound 16e, yielding 70 mg (15%
yield) of a white solid; m/z 605 [M+H].sup.+. .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. ppm 1.08-1.20 (m, 1H) 1.22-1.79 (m, 13H) 1.88
(s, 6H) 2.40-2.47 (m, 1H) 2.69-2.83 (m, 1H) 2.92-3.14 (m, 4H)
3.56-3.72 (m, 1H) 3.89 (s, 3H) 3.92-4.04 (m, 1H) 4.26 (d, J=14.67
Hz, 1H) 4.86 (d, J=14.09 Hz, 1H) 7.18 (dd, J=8.61, 2.15 Hz, 1H)
7.22 (d, J=2.15 Hz, 1H) 7.46-7.57 (m, 2H) 7.80-7.92 (m, 1H) 8.48
(s, 1H) 11.39 (br. s., 1H)
Example 18
Synthesis of Compound 18
##STR00142##
[0323] The synthesis of the title compound 18 was performed
following the 4-step procedure reported for the synthesis of
compound 17, starting from intermediate 1b instead of 16b, and
yielding 0.5 g of a white solid; m/z 591 [M+H].sup.+. .sup.1H NMR
(400 MHz, DMSO-d.sub.6) .delta. ppm 1.01-1.19 (m, 1H) 1.18-1.52 (m,
5H) 1.54-1.79 (m, 4H) 1.80-2.08 (m, 4H) 2.42-2.48 (m, 1H) 2.63-2.80
(m, 1H) 2.93 (s, 3H) 2.98-3.14 (m, 1H) 3.43-3.75 (m, 5H) 3.85 (s,
3H) 4.43 (d, J=14.87 Hz, 1H) 5.04 (d, J=14.48 Hz, 1H) 6.84 (br. s.,
1H) 7.09 (s, 1H) 7.18 (d, J=8.22 Hz, 1H) 7.45 (d, J=8.22 Hz, 1H)
7.55 (d, J=8.41 Hz, 1H) 7.87 (d, J=8.41 Hz, 1H) 8.35 (br. s., 1H)
11.33 (br. s., 1H)
Example 19
Synthesis of Compound 19
##STR00143##
[0325] The synthesis of the title compound 19 was performed
following the 5-step procedure reported for the synthesis of
compound 1, starting from intermediate 10-tert-butyl 6-methyl
13-cyclohexyl-3-fluoro-7H-indolo[2,1-a][2]benzazepine-6,10-dicarboxylate
19a instead of 10-tert-butyl 6-methyl
13-cyclohexyl-3-methoxy-7H-indolo[2,1-a][2]benzazepine-6,10-dicarboxylate
1a, and yielded 180 mg of a white solid; m/z 595 [M+H].sup.+.
.sup.1H NMR (400 MHz, CHLOROFORM-d) .delta. ppm 1.11-1.29 (m, 1H)
1.29-1.53 (m, 3H) 1.67-1.83 (m, 3H) 1.87-2.11 (m, 4H) 2.30 (br. s.,
3H) 2.69-2.82 (m, 1H) 2.81-2.98 (m, 1H) 3.11 (s, 3H) 3.46-3.58 (m,
1H) 3.59-3.79 (m, 3H) 3.90-4.08 (m, 1H) 4.24-4.38 (m, 1H) 4.43 (dd,
J=14.73, 1.27 Hz, 1H) 4.97 (d, J=14.63 Hz, 1H) 6.73 (s, 1H) 7.11
(dd, J=9.27, 2.63 Hz, 1H) 7.17-7.30 (m, 1H) 7.57 (dd, J=8.68, 5.76
Hz, 1H) 7.69 (s, 1H) 7.67 (dd, J=8.78, 1.56 Hz, 1H) 7.90 (d, J=8.78
Hz, 1H) 9.84 (br. s., 1H)
Example 20
Synthesis of Compound 20
##STR00144##
[0327] The synthesis of the title compound 20 was performed
following the 5-step procedure reported for the synthesis of
compound 1, starting from intermediate 10-tert-butyl 6-methyl
13-cyclohexyl-3-fluoro-5-methyl-7H-indolo[2,1-a][2]benzazepine-6,10-dicar-
boxylate 20a instead of 10-tert-butyl 6-methyl
13-cyclohexyl-3-methoxy-7H-indolo[2,1-a][2]benzazepine-6,10-dicarboxylate
1a, and yielded 130 mg of a white solid; m/z 609 [M+H].sup.+.
.sup.1H NMR (400 MHz, CHLOROFORM-d) .delta. ppm 1.14-1.31 (m, 1H)
1.32-1.46 (m, 3H) 1.64-1.81 (m, 3H) 1.84 (s, 3H) 1.87-1.99 (m, 3H)
2.01 (s, 3H) 2.47 (d, J=14.63 Hz, 1H) 2.73-2.87 (m, 1H) 3.14 (s,
3H) 3.43 (d, J=15.02 Hz, 1H) 3.56-3.64 (m, 2H) 3.65 (d, J=3.12 Hz,
1H) 3.74-3.88 (m, 1H) 4.00-4.12 (m, 1H) 4.35 (d, J=14.83 Hz, 1H)
4.64-4.75 (m, 1H) 4.81 (d, J=14.63 Hz, 1H) 7.16-7.32 (m, 2H) 7.53
(dd, J=8.39, 6.05 Hz, 1H) 7.64 (s, 1H) 7.70 (d, J=8.39 Hz, 1H) 7.91
(d, J=8.39 Hz, 1H) 10.09 (br. s., 1H)
Example 21
Synthesis of Compound 21
##STR00145##
[0329] The synthesis of the title compound 21 was performed
following the 5-step procedure reported for the synthesis of
compound 1, starting from intermediate 10-tert-butyl 6-methyl
3-chloro-13-cyclohexyl-7H-indolo[2,1-a][2]benzazepine-6,10-dicarboxylate
21a instead of 10-tert-butyl 6-methyl
13-cyclohexyl-3-methoxy-7H-indolo[2,1-a][2]benzazepine-6,10-dicarboxylate
1a, and yielding 270 mg of a white solid; m/z 611 [M+H].sup.+.
.sup.1H NMR (400 MHz, DMSO-d6) .delta. ppm 1.08-1.22 (m, 1H)
1.31-1.52 (m, 3H) 1.63-1.78 (m, 2H) 1.81-2.09 (m, 4H) 2.50 (s, 3H)
2.69-2.80 (m, 1H) 3.00 (s, 3H) 3.08-3.19 (m, 1H) 3.19-3.28 (m, 1H)
3.46-3.88 (m, 6H) 4.52 (d, J=14.87 Hz, 1H) 5.12 (d, J=13.11 Hz, 1H)
6.97 (s, 1H) 7.49 (d, J=7.83 Hz, 1H) 7.58-7.70 (m, 3H) 7.94 (d,
J=8.61 Hz, 1H) 8.36 (s, 1H) 11.39 (br. s., 1H)
Example 22
Synthesis of Compound 22
##STR00146##
[0331] The synthesis of the title compound 22 was performed
following the 5-step procedure reported for the synthesis of
compound 1, using N.sup.1,N.sup.6-dimethylhexane-1,6-diamine
instead of 2,2'-oxybis(N-methylethanamine) in step 2, and yielded
50 mg of a white solid; m/z 619 [M+H].sup.+. .sup.1H NMR (400 MHz,
CHLOROFORM-d) .delta. ppm 1.00-1.64 (m, 11H) 1.66-1.87 (m, 3H)
1.87-2.15 (m, 4H) 2.47 (s, 3H) 2.66-2.91 (m, 2H) 3.23 (s, 3H)
3.25-3.33 (m, 1H) 3.33-3.45 (m, 1H) 3.90 (s, 3H) 4.09-4.25 (m, 1H)
4.39 (d, J=14.28 Hz, 1H) 5.14 (d, J=14.48 Hz, 1H) 6.81 (s, 1H) 6.90
(s, 1H) 7.06 (dd, J=8.61, 2.15 Hz, 1H) 7.45 (d, J=8.22 Hz, 1H) 7.50
(d, J=8.61 Hz, 1H) 7.81-7.96 (m, 2H) 8.94 (br. s., 1H)
Example 23
Synthesis of Compound 23
##STR00147##
[0333] The synthesis of the title compound 23 was performed
following the 5-step procedure reported for the synthesis of
compound 1, using
N.sup.1,N.sup.2-dimethyl-N.sup.1-(2-(methylamino)ethyl)ethane-1,2-diamine
instead of 2,2'-oxybis(N-methylethanamine) in step 2, and yielded
20 mg of a white solid; m/z 592 [M+H].sup.+. .sup.1H NMR (400 MHz,
DMSO-d6) .delta. ppm 1.04 (d, J=5.87 Hz, 1H) 1.06-1.22 (m, 1H)
1.27-1.51 (m, 3H) 1.60-1.78 (m, 2H) 1.80-1.92 (m, 1H) 1.92-2.07 (m,
3H) 2.12 (s, 3H) 2.27-2.41 (m, 1H) 2.69-2.83 (m, 2H) 2.83-2.97 (m,
2H) 3.01-3.15 (m, 2H) 3.17-3.28 (m, 2H) 3.86 (s, 3H) 4.21 (d,
J=15.65 Hz, 1H) 5.54 (d, J=15.65 Hz, 1H) 7.11-7.25 (m, 2H) 7.35 (s,
1H) 7.47 (d, J=8.22 Hz, 1H) 7.53 (d, J=9.00 Hz, 1H) 7.70-7.83 (m,
1H) 8.32 (br. s., 1H) 8.37-8.50 (m, 1H)
Example 24
Synthesis of Compound 24
##STR00148##
[0335] The synthesis of the title compound 24 was performed
following the 4-step procedure reported for the synthesis of
compound 17, starting from intermediate
10-(tert-butoxycarbonyl)-2-chloro-13-cyclohexyl-7H-indolo[2,1-a][2]benzaz-
epine-6-carboxylic acid 24b instead of
10-(tert-butoxycarbonyl)-13-cyclohexyl-3-methoxy-5-methyl-7H-indolo[2,1-a-
][2]benzazepine-6-carboxylic acid 16b, and yielded 0.25 g of a
white solid; m/z 595 [M+H].sup.+. .sup.1H NMR (400 MHz,
Chloroform-d) .delta. ppm 1.25-1.5 (m, 4H) 1.5-1.8 (m, 4H) 1.9-2.1
(m, 4H) 1.8 (s., 3H) 2.8-2.13 (m, 3H) 2.5-2.6 (m, 2H) 3.2 (s, 3H)
3.6 (br. s., 1H) 4.1 (br. s., 1H) 4.45 (d, J=15 Hz, 1H) 5 (d, J=15
Hz, 1H) 6.6 (s, 1H) 7.25 (d, J=8.4 Hz, 1H) 7.4 (dd, J=8.5, J=2.5
Hz, 1H) 7.5-7.6 (m, 2H) 7.69 (s, 1H) 7.9 (d, J=8.4 Hz, 1H) 9.1 (br.
s., 1H)
Example 25
Synthesis of Compound 25
##STR00149##
[0337] The synthesis of the title compound 25 was performed
following the 5-step procedure reported for the synthesis of
compound 10, starting from intermediate 10-tert-butyl 6-methyl
13-cyclohexyl-3-methoxy-7H-indolo[2,1-a][2]benzazepine-6,10-dicarboxylate
1a instead of 5-tert-butyl 1a-methyl
8-cyclohexyl-11-methoxy-1,12b-dihydrocyclopropa[d]indolo[2,1-a][2]benzaze-
pine-1a,5(2H)-dicarboxylate 8a, and yielded 45 mg of a white solid;
m/z 577[M+H].sup.+. .sup.1H NMR (400 MHz, DMSO-d6) .delta. ppm
1.03-1.19 (m, 1H) 1.25-1.49 (m, 4H) 1.49-2.29 (m, 10H) 2.67-2.82
(m, 1H) 2.84-3.04 (m, 1H) 3.05-3.24 (m, 1H) 3.48-3.72 (m, 5H) 3.86
(s, 3H) 4.42 (d, J=14.67 Hz, 1H) 5.00 (d, J=14.28 Hz, 1H) 6.84 (br.
s., 1H) 7.09 (s, 1H) 7.18 (d, J=8.41 Hz, 1H) 7.47 (d, J=7.83 Hz,
1H) 7.55 (d, J=8.41 Hz, 1H) 7.75-7.92 (m, 1H) 8.19-8.41 (m, 1H)
11.27 (br. s., 1H)
Example 26
Synthesis of Compound 26
##STR00150##
[0338] Step 1
##STR00151##
[0340] A solution of 606 mg (1.24 mmole) of 1b, 410 mg (1.1 eq) of
26a, 710 mg (1.5 eq) of HATU and 0.65 mL (3 eq) of diisopropylethyl
amine in dry DMF (10 mL) was stirred at RT during 1 h. The RM was
then diluted with water and the resulting yellow precipitate was
filtered off, washed with water, and purified by flash
chromatography (eluant DCM to DCM/MeOH 0.5%) to give a quantitative
yield of the desired product 26b as a yellow powder; m/z 771
[M+H].sup.+.
Step 2
##STR00152##
[0342] To a solution of 1.1 g (1.44 mmole) of 26b and thiophenol
(0.32 g, 2 eq) in dry DMF (15 mL) was added cesium carbonate (0.94
g, 2 eq) at RT. After 2 h, the RM was diluted with water and
extracted with EtOAc. The organic layer was washed with brine,
dried over magnesium sulfate, filtered and concentrated. The
resulting residue was further purified by flash chromatography
(eluant: DCM to DCM/NH3 in MeOH 85/15) to give 0.77 g (90% yield)
of 26c as a yellow powder; m/z 586 [M+H].sup.+.
Step 3
##STR00153##
[0344] A mixture of 26c (0.72 g, 1.23 mmole) and sulfamide (0.35 g,
3 eq) in dioxane (15 mL) was refluxed until completion (.about.7
h). The RM was then concentrated under vacuo and the residue was
triturated in DCM. The resulting precipitate of sulfamide in excess
was filtered off. The organic layer was concentrated and purified
by flash chromatography (eluant: DCM to DCM/MeOH 1%) to give 776 mg
(95% yield) of the desired product 26d as a light yellow powder;
m/z 665 [M+H].sup.+.
Step 4
##STR00154##
[0346] A solution of 26d (0.72 g, 1.086 mmole) in 10 mL of HCl in
isopropanol and 5 mL of DCM was stirred at RT for 3 h. The RM was
then concentrated under vacuo, and the residue was triturated in
diethyl ether. The resulting precipitate was filtered off, washed
with ether and dried in a vacuum oven overnight to give 661 mg (97%
yield) of the desired product 26e as a light yellow powder; m/z 609
[M+H].sup.+.
Step 5
##STR00155##
[0348] A solution of 26e (0.6 g, 0.971 mmole) and CDI (0.205 g, 1.3
eq) in acetonitrile (10 mL) was heated at 60.degree. C. until
complete formation of the acyl imidazole intermediate (.about.1 h).
The RM was then diluted with 20 mL of acetonitrile and DBU (0.296
g, 2 eq) was added at RT. The RM was stirred at RT until
completion, then was concentrated. The residue was redissolved in
water and acetic acid was then added until pH 2. The resulting
precipitate was filtered off, washed with water, and purified by
flash chromatography (eluant: DCM to DCM/MeOH 5%) to give 0.315 g
(55% yield) of the desired product 26 as a slightly yellow powder;
m/z 591 [M+H].sup.+. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta.
ppm 1.07-2.09 (m, 16H) 2.71-2.84 (m, 1H) 2.94 (s, 3H) 3.01-3.18 (m,
2H) 3.19-3.31 (m, 2H) 3.87 (s, 3H) 4.25 (d, J=15.06 Hz, 1H) 5.52
(d, J=15.26 Hz, 1H) 7.16-7.26 (m, 2H) 7.32-7.44 (m, 2H) 7.54 (d,
J=9.19 Hz, 1H) 7.86 (d, J=8.61 Hz, 1H) 8.26 (s, 1H) 8.40-8.51 (m,
1H) 11.61 (br. s., 1H)
Example 27
Synthesis of Compound 27
##STR00156##
[0350] The synthesis of the title compound 27 was performed
following the 5-step procedure reported for the synthesis of
compound 26, starting from intermediate
10-(tert-butoxycarbonyl)-2-chloro-13-cyclohexyl-7H-indolo[2,1-a][2]benzaz-
epine-6-carboxylic acid 24b instead of
10-(tert-butoxycarbonyl)-13-cyclohexyl-3-methoxy-7H-indolo[2,1-a][2]benza-
zepine-6-carboxylic acid 1b, and yielded 85 mg of a yellow solid;
m/z 596 [M+H].sup.+. .sup.1H NMR (400 MHz, Chloroform-d) .delta.
ppm 1.21-1.5 (m, 10H) 1.75-1.8 (m, 2H) 1.9-2.1 (m, 4H) 2.75 (br.
s., 1H) 3.01 (s, 3H) 3.1-3.2 (m, 2H) 3.5-3.6 (m, 2H) 4.23 (dd,
J=15.28, 1.27 Hz, 1H) 5.6 (d, J=15.28 Hz, 1H) 7.4 (s, 1H) 7.5-7.6
(m, 3H) 7.65 (d, J=8.5 Hz, 1H) 7.8 (s, 1H) 7.9 (d, J=8.5 Hz, 1H)
7.69 (s, 1H) 8.64 (br. s., 1H)
Example 28
Synthesis of Compound 28
##STR00157##
[0352] The synthesis of the title compound 28 was performed
following the 5-step procedure reported for the synthesis of
compound 10, starting from intermediate 10-tert-butyl 6-methyl
13-cyclohexyl-3-methoxy-7H-indolo[2,1-a][2]benzazepine-6,10-dicarboxylate
1a instead of 5-tert-butyl 1a-methyl
8-cyclohexyl-11-methoxy-1,12b-dihydrocyclopropa[d]indolo[2,1-a][2]benzaze-
pine-1a,5(2H)-dicarboxylate 8a, and using
N-[4-(methylamino)butyl]-N-(1-methylethyl)sulfamide 28a instead of
N-(4-aminobutyl)-N-methylsulfamide 10b, and yielded 50 mg of the
desired product 28; m/z 619 [M+H].sup.+. .sup.1H NMR (400 MHz,
CHLOROFORM-d) .delta. ppm 1.05-1.15 (m, 1H) 1.18 (d, J=6.65 Hz, 3H)
1.25 (d, J=6.46 Hz, 3H) 1.28-1.51 (m, 4H) 1.53-2.31 (m, 13H)
2.67-2.85 (m, 1H) 3.01-3.19 (m, 1H) 3.51-3.73 (m, 1H) 3.89 (s, 3H)
3.95-4.15 (m, 1H) 4.42 (d, J=14.48 Hz, 1H) 4.52-4.72 (m, 1H) 5.01
(d, J=14.48 Hz, 1H) 6.68 (s, 1H) 6.87 (s, 1H) 7.05 (d, J=8.41 Hz,
1H) 7.52 (d, J=8.41 Hz, 1H) 7.63 (d, J=8.22 Hz, 1H) 7.77-7.99 (m,
2H) 9.42 (br. s., 1H)
Example 29
Synthesis of Compound 10b
##STR00158##
[0354] A mixture of tert-butyl 4-(methylamino)butylcarbamate (4 g,
19.77 mmoles) and sulfuric diamide (7.6 g, 4 eq) in dioxane (10 mL)
was heated at 100.degree. C. in a microwave oven during 30 minutes.
The RM was then concentrated in vacuo, and DCM was added. The
resulting white precipitate of sulfuric diamide in excess was
filtered off, and the filtrate was successively washed with diluted
HCl, then brine, dried over magnesium sulfate, filtered and
concentrated. Trituration in diisopropyl ether afforded 3.55 g (64%
yield) of tert-butyl 4-(methyl(sulfamoyl)amino)butylcarbamate 10b
as a white solid; m/z 282 [M+H].sup.+.
Example 30
Synthesis of Compound 26a
##STR00159##
[0355] Step 1
##STR00160##
[0357] To a solution of tert-butyl 5-aminopentylcarbamate 30a (20
g, 99 mmoles) and 2-nitrobenzene-1-sulfonyl chloride 30b (23 g,
1.05 eq) in DCM (200 mL) was added dropwise diisopropyl ethyl amine
(19.2 g, 1.5 eq) at 0.degree. C. After stirring at RT overnight,
the RM was successively washed with an aqueous solution of citric
acid, then brine, dried over magnesium sulfate, filtered and
concentrated. Trituration in diisopropyl ether afforded 32.61 g
(85% yield) of tert-butyl
5-(2-nitrophenylsulfonamido)pentylcarbamate 30c as a white solid;
m/z 388 [M+H].sup.+.
Step 2
##STR00161##
[0359] To a mixture of tert-butyl
5-(2-nitrophenylsulfonamido)pentylcarbamate 30c (32.61 g, 84
mmoles) and potassium carbonate (13.96 g, 1.2 eq) in acetone (300
mL) was added methyl iodide (5.5 mL, 1.05 eq). After stirring at RT
overnight, more methyl iodide (1 eq) and potassium carbonate (0.6
eq) was added and the RM was stirred at RT until completion. The RM
was then diluted with water and extracted with DCM. The organic
layer was separated, washed with brine, dried over magnesium
sulfate, filtered and concentrated. Trituration in diisopropyl
ether afforded 31.59 g (93% yield) of tert-butyl
5-(N-methyl-2-nitrophenylsulfonamido)pentylcarbamate 30d as a white
solid; m/z 402 [M+H].sup.+.
Step 3
##STR00162##
[0361] A solution of tert-butyl
5-(N-methyl-2-nitrophenylsulfonamido)pentylcarbamate 30d (31.5 g,
79 mmoles) and trifluoro acetic acid (29.2 mL, 5 eq) in DCM (300
mL) was stirred at RT until completion (.about.16 h). The RM was
then concentrated under vacuo, redissolved in DCM, washed with a
saturated sodium bicarbonate aqueous solution (2 times), then
brine, dried over magnesium sulfate, filtered and concentrated.
Trituration in diisopropyl ether afforded 23.7 g (quantitative
yield) of N-(5-aminopentyl)-N-methyl-2-nitrobenzenesulfonamide 26a
as a slightly yellow solid; m/z 302 [M+H].sup.+.
Example 31
Synthesis of Compound 28a
##STR00163##
[0362] Step 1
##STR00164##
[0364] A mixture of tert-butyl 4-aminobutyl(methyl)carbamate 31a
(287 mg, 1.42 mmole), acetone (75 mg, 1.29 mmole) and sodium
triacetoxyborohydride (383 mg, 1.8 mmole) was stirred under
nitrogen at RT until completion. The RM was then concentrated,
diluted with a saturated sodium bicarbonate aqueous solution, and
extracted with ether (2 times). The organic layers were combined,
dried over magnesium sulfate, filtered and concentrated to give 200
mg (63% yield) of the desired product tert-butyl
4-(isopropylamino)butyl(methyl)carbamate 31b, used without further
purification in the next step; m/z 245 [M+H].sup.+.
Step 2
##STR00165##
[0366] A mixture of tert-butyl
4-(isopropylamino)butyl(methyl)carbamate 31b (3.38 g, 13.8 mmoles)
and sulfuric diamide (3.99 g, 3 eq) in dioxane (10 mL) was heated
at 110.degree. C. in a microwave oven during 60 minutes. The RM was
then concentrated in vacuo, and DCM was added. The resulting white
precipitate of sulfuric diamide in excess was filtered off, and the
filtrate was concentrated in vacuo. The residue was purified by
flash chromatography (eluant: DCM to DCM/MeOH 20%) to give 1.7 g
(38% yield) of the desired product tert-butyl
4-(isopropyl(sulfamoyl)amino)butyl(methyl)carbamate 28a; m/z 324
[M+H].sup.+.
Example 32
Synthesis of Compound 19a
##STR00166##
[0367] Step 1
##STR00167##
[0369] A mixture of tert-butyl
2-bromo-3-cyclohexyl-1H-indole-6-carboxylate 32a (5 g, 13.22
mmoles, synthesized as described in US 2007270406 A1),
pinacolborane (5.75 mL, 3 eq) and triethylamine (7.35 mL, 4 eq) in
THF (50 mL) was stirred at RT during 3 h. Palladium acetate (90 mg,
0.03 eq) and biphenyl-2-yldicyclohexylphosphine (556 mg, 0.12 eq)
were then added and the RM was heated at 80.degree. C. during 2 h.
The reaction mixture was then allowed to cool down to RT and poured
in a solution of watered NH.sub.4Cl then extracted with ethyl
acetate. The organic layers were dried over magnesium sulfate,
filtered and concentrated. The residue was purified by column
chromatography using a gradient of ethyl acetate in heptane to give
3.5 g (70% yield) of the desired product tert-butyl
3-cyclohexyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole-6--
carboxylate 32b; m/z 426 [M+H].sup.+.
Step 2
##STR00168##
[0371] To a mixture of tert-butyl
3-cyclohexyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole-6--
carboxylate 32b (2.77 g, 6.5 mmoles) and
2-bromo-5-fluorobenzaldehyde 32c (1.58 g, 1.2 eq) in DME (40 mL)
was added a solution of sodium carbonate (2.07 g, 3 eq) in water
(15 mL). The resulting mixture was then flushed with nitrogen at RT
during 10 minutes. After the addition of palladium tetrakis
triphenylphospine (376 mg, 0.05 eq), the RM was heated at
70.degree. C. during 1 h. The mixture was then allowed to cool down
to RT and poured in water, then extracted with ethyl acetate (3
times). The organic layers were combined, dried over MgSO.sub.4,
filtered and concentrated. The residue was recrystallized from
di-isopropyl ether/heptane to give 2 g (73% yield) of the desired
product tert-butyl
3-cyclohexyl-2-(4-fluoro-2-formylphenyl)-1H-indole-6-carboxylate
32d as a white solid; m/z 422 [M+H].sup.+.
Step 3
##STR00169##
[0373] A mixture of tert-butyl
3-cyclohexyl-2-(4-fluoro-2-formylphenyl)-1H-indole-6-carboxylate
32d (2 g, 4.75 mmoles), cesium carbonate (1.85 g, 1.2 eq) and
methyl 2-(dimethoxyphosphoryl)acrylate (16.475 mL, 0.36 M solution
in toluene, 1.25 eq) in DMF (80 mL) was stirred at 60.degree. C.
during 2 h. The reaction mixture was then allowed to cool down to
room temperature, poured in water and extracted with ethyl acetate.
The organic layer was then dried over MgSO.sub.4, filtered and
concentrated. The residue was purified by column chromatography
using heptanes/dichloromethane to give 2 g (86% yield) of the
desired product 10-tert-butyl 6-methyl
13-cyclohexyl-3-fluoro-7H-indolo[2,1-a][2]benzazepine-6,10-dicarboxylate
19a; m/z 490 [M+H].sup.+.
Example 33
Synthesis of Compound 20a
##STR00170##
[0374] Step 1
##STR00171##
[0376] To a solution of 2-bromo-5-fluoro-benzonitrile 33a (10 g, 50
mmol) in dry tetrahydrofuran (100 mL) under nitrogen was added
methylmagnesium bromide (3.2 M in ether, 19 mL, 60.0 mmol), and the
resulting mixture was heated to reflux for 4 hours. The RM was then
cooled down to RT, poured into a 2 N HCl solution (100 mL) and then
diluted with methanol (100 mL). The resulting green solution was
concentrated on a steam bath for 1 h at which point the organic
solvents had been removed and the crude product had precipitated.
The reaction mixture was then extracted with ethyl acetate, dried
over MgSO.sub.4 and concentrated. The residue was purified by
column chromatography using heptane and dichloromethane to give
4.88 g (45% yield) of the desired product
1-(2-bromo-5-fluorophenyl)ethanone 33b as a pink oil; m/z 218
[M+H].sup.+.
Step 2
##STR00172##
[0378] The title product tert-butyl
2-(2-acetyl-4-fluorophenyl)-3-cyclohexyl-1H-indole-6-carboxylate
33c was synthesized following the procedure reported for the
synthesis of tert-butyl
3-cyclohexyl-2-(4-fluoro-2-formylphenyl)-1H-indole-6-carboxylate
32d, using 1-(2-bromo-5-fluorophenyl)ethanone 33b instead of
2-bromo-5-fluorobenzaldehyde 32c, and was obtained in 65% yield as
a white solid; m/z 436 [M+H].sup.+.
Step 3
##STR00173##
[0380] The title product 10-tert-butyl 6-methyl
13-cyclohexyl-3-fluoro-5-methyl-7H-indolo[2,1-a][2]benzazepine-6,10-dicar-
boxylate 20a was synthesized following the procedure reported for
the synthesis of 10-tert-butyl 6-methyl
13-cyclohexyl-3-fluoro-7H-indolo[2,1-a][2]benzazepine-6,10-dicarboxylate
19a, using tert-butyl
2-(2-acetyl-4-fluorophenyl)-3-cyclohexyl-1H-indole-6-carboxylate
33c instead of tert-butyl
3-cyclohexyl-2-(4-fluoro-2-formylphenyl)-1H-indole-6-carboxylate
32d, and was obtained in 11% yield as a white solid; m/z 504
[M+H].sup.+.
Example 34
Synthesis of Compound 21a
##STR00174##
[0381] Step 1
##STR00175##
[0383] The bromo indole derivative 32a (5 g, 13.22 mmol),
4-chloro-2-formylphenylboronic acid 34a (3.17 g, 17.18 mmol) and
potassium carbonate (4.20 g, 30.4 mmol) were dissolved in 100 mL of
1,2-dimethoxyethane (80 ml)/water (20 ml) 4/1 and the obtained
solution was flushed thoroughly with argon. Then
bis(triphenylphosphine)palladium(II) chloride (0.464 g, 0.661 mmol)
was added and the reaction was heated to 63.degree. C. under argon
during 3 h. The reaction was then diluted with EtOAc, washed with
water and with sat. aq. NaHCO.sub.3, dried (brine, sulfate) and
evaporated. The residue was stripped with DIPE and stirred and
sonicated in heptane with a few mL DIPE added. The solid was
filtered off and dried to afford 4.97 g (86% yield) of the desired
product tert-butyl
2-(4-chloro-2-formylphenyl)-3-cyclohexyl-1H-indole-6-carboxylate
34b; m/z 437 [M+H].sup.+.
Step 2
##STR00176##
[0385] The indole derivative 34b (4.95 g, 11.30 mmol) and cesium
carbonate (4.42 g, 13.56 mmol) were dissolved in
N,N-dimethylformamide (dry) (50 ml) and methyl
2-(dimethoxyphosphoryl)acrylate (3.23 g, 14.13 mmol) was added. The
RM was stirred at 65.degree. C. during 2 h. It was then cooled to
rt and dropped onto 300 ml of water vigorously stirred. The
resulting yellowish solid was filtered off, washed with water and
dried to afford 5.40 g (94% yield) of the desired product
10-tert-butyl 6-methyl
3-chloro-13-cyclohexyl-7H-indolo[2,1-a][2]benzazepine-6,10-dicarboxylate
21a, used without further purification in the next step; m/z 507
[M+H].sup.+.
Example 35
Synthesis of Compound 24a
##STR00177##
[0387] The title compound 24a was synthesized following the 2-step
procedure reported for the synthesis of 10-tert-butyl 6-methyl
3-chloro-13-cyclohexyl-7H-indolo[2,1-a][2]benzazepine-6,10-dicarboxylate
21a, using 5-chloro-2-formylphenylboronic acid in the first step,
instead of 4-chloro-2-formylphenylboronic acid 34a, and was
obtained with an overall yield of 70% as a yellowish solid; m/z 507
[M+H].sup.+.
Example 36
Synthesis of Compound 24b
##STR00178##
[0389] A solution of NaOH (6.38 g) in 25 mL of water was added to a
stirred solution of the indole derivative 24a in THF (100 mL) and
MeOH (150 mL). After 1 hour the reaction was concentrated under
reduced pressure, then diluted with ice-cold water (150 mL). The pH
of the resulting solution was adjusted to 6 with HCl then extracted
with dichloromethane and dried over MgSO.sub.4. The solvent was
removed then the residue was purified by column chromatography
using DCM/MeOH as eluant to give 1.7 g (87% yield) of a yellowish
solid; m/z 492 [M+H].sup.+.
Example 37
Synthesis of Compound 16a
##STR00179##
[0390] Step 1
##STR00180##
[0392] Ethane-1,2-diol (4.06 g) and Tos-OH (0.41 g) were added to a
solution of 1-(2-bromo-5-methoxyphenyl)ethanone 37a (5 g) in
toluene (950 ml). The solution was heated under reflux with
stirring in a 3-neck round-bottomed flask fitted with a Dean-Stark
receiver for 3 hours. The reaction mixture was then cooled to room
temperature. The mixture was transferred to a separating funnel and
a sodium carbonate solution (1 M, 50 ml) was added. The mixture was
agitated and two phases formed. The organic layer was separated,
washed with water (2.times.50 ml), dried over MgSO.sub.4 and
concentrated under vacuum to afford 6.5 g (quantitative yield) of
the desired product
2-(2-bromo-5-methoxyphenyl)-2-methyl-1,3-dioxolane 37b as a white
solid.
Step 2
##STR00181##
[0394] The bromo derivative 37b (6 g) was dissolved in dry THF (60
ml) and the solution was cooled down to -78.degree. C. Then n-BuLi
(16.5 ml) was added carefully at such a rate that the temperature
did not exceed -60.degree. C. After 1 h, B(O-i-Pr).sub.3 (6.2 g)
was added neatly dropwise at -78.degree. C. After all was added,
the cooling bath was removed. The mixture was stirred at 0.degree.
C. for 2.5 h, then 2 N HCl (60 ml) was added, and the RM stirred at
r.t. for 2 h. The organic solvent was then removed under vacuum and
the aqueous layer was saturated with NaCl and extracted with EtOAc.
The organic layer was dried over Na.sub.2SO.sub.4 and evaporated
under vacuum to give 3 g of the desired product
2-acetyl-4-methoxyphenylboronic acid 37c.
Step 3
##STR00182##
[0396] The title product tert-butyl
2-(2-acetyl-4-methoxyphenyl)-3-cyclohexyl-1H-indole-6-carboxylate
37d was synthesized by following a similar procedure to that used
for the synthesis of tert-butyl
2-(4-chloro-2-formylphenyl)-3-cyclohexyl-1H-indole-6-carboxylate
34b, using 2-acetyl-4-methoxyphenylboronic acid 37c instead of
4-chloro-2-formylphenylboronic acid 34a.
Step 4
##STR00183##
[0398] The title product 10-tert-butyl 6-methyl
13-cyclohexyl-3-methoxy-5-methyl-7H-indolo[2,1-a][2]benzazepine-6,10-dica-
rboxylate 16a was synthesized by following a similar procedure to
that used for the synthesis of 10-tert-butyl 6-methyl
13-cyclohexyl-3-fluoro-5-methyl-7H-indolo[2,1-a][2]benzazepine-6,10-dicar-
boxylate 20a, using tert-butyl
2-(2-acetyl-4-methoxyphenyl)-3-cyclohexyl-1H-indole-6-carboxylate
37d instead of tert-butyl
2-(2-acetyl-4-fluorophenyl)-3-cyclohexyl-1H-indole-6-carboxylate
33c.
Example 38
Synthesis of Compound 38
##STR00184##
[0400] The synthesis of the title compound 38 was performed
following the 5-step procedure reported for the synthesis of
compound 10, starting from intermediate
13-cyclohexyl-3-methoxy-7H-benzo[3,4]azepino[1,2-a]indole-6,10-dicarboxyl-
ic acid 10-tert-butyl ester 6-methyl ester 1a instead of
5-tert-butyl 1a-methyl
8-cyclohexyl-11-methoxy-1,12b-dihydrocyclopropa[d]indolo[2,1-a]-
[2]benzazepine-1a,5(2H)-dicarboxylate 8a, and yielded 60 mg of a
beige solid; m/z 577 [M+H].sup.+.
Example 39
Activity of Compounds of Formula (I)
Replicon Assay
[0401] The compounds of formula (I) were examined for activity in
the inhibition of HCV RNA replication in a cellular assay. The
assay demonstrated that the compounds of formula (I) inhibited a
HCV functional cellular replicating cell line, also known as HCV
replicons. The cellular assay was based on a bicistronic expression
construct, as described by Lohmann et al. (1999) Science vol. 285
pp. 110-113 with modifications described by Krieger et al. (2001)
Journal of Virology 75: 4614-4624, in a multi-target screening
strategy. In essence, the method was as follows.
[0402] The assay utilized the stably transfected cell line Huh-7
luc/neo (hereafter referred to as Huh-Luc). This cell line harbors
an RNA encoding a bicistronic expression construct comprising the
wild type NS3-NS5B regions of HCV type 1b translated from an
Internal Ribosome Entry Site (IRES) from encephalomyocarditis virus
(EMCV), preceded by a reporter portion (FfL-luciferase), and a
selectable marker portion (neo.sup.R, neomycine
phosphotransferase). The construct is bordered by 5' and 3' NTRs
(non-translated regions) from HCV type 1b. Continued culture of the
replicon cells in the presence of G418 (neo.sup.R) is dependent on
the replication of the HCV RNA. The stably transfected replicon
cells that express HCV RNA, which replicates autonomously and to
high levels, encoding inter alia luciferase, are used for screening
the antiviral compounds.
[0403] The replicon cells were plated in 384 well plates in the
presence of the test and control compounds which were added in
various concentrations. Following an incubation of three days, HCV
replication was measured by assaying luciferase activity (using
standard luciferase assay substrates and reagents and a Perkin
Elmer ViewLux.TM. ultraHTS microplate imager). Replicon cells in
the control cultures have high luciferase expression in the absence
of any inhibitor. The inhibitory activity of the compound was
monitored on the Huh-Luc cells, enabling a dose-response curve to
be generated for each test compound. EC50 values were then
calculated, which value represents the amount of the compound
required to decrease by 50% the level of detected luciferase
activity, or more specifically, the ability of the genetically
linked HCV replicon RNA to replicate.
Enzymatic Assay
1. HCV NS5B 1bJ4
1.a) Protein Purification
[0404] The cDNA encoding NS5B amino acid 1-570 (HC-J4, genotype 1b,
pCV-J4L6S, genebank accession number AF054247) was subcloned into
the Nhe I and Xho I restriction sites of pET-21b. Expression of the
subsequent His-tagged C-terminal 21 amino acid deleted NS5B was
performed as follows:
[0405] The NS5B expression construct was transformed into E. coli
BL21(DE3) (Novagen, Madison, Wis.). Five milliliter of LB-medium
supplemented with ampicillin (50 .mu.g/mL) was inoculated with one
colony. When the pre-culture reached an optical density of 0.6
measured at 600 nm, it was transferred to fresh LB-medium
supplemented with ampicillin, at a ratio of 1:200. Cells were grown
to an optical density at 600 nm of 0.6, after which the expression
cultures were shifted to a growth temperature of 20.degree. C.
following induction with
ispopropyl-1-thio-.beta.-D-galactopyranoside and MgCl.sub.2 at a
final concentration of 0.4 mM and 10 .mu.M, respectively. After 10
h of induction, cells were harvested by centrifugation and
resuspended in 20 mM Tris-HCl, pH 7.5, 300 mM NaCl, 10% glycerol,
0.1% NP40, 4 mM MgCl.sub.2, 5 mM DTT supplemented with EDTA-free
Complete Protease Inhibitor (Roche, Basel, Switzerland). Cell
suspensions were disrupted by sonication and incubated with 10-15
mg/L of DNase I (Roche, Basel, Switzerland) for 30 min. Cell debris
was removed through ultracentrifugation at 30,000.times.g for 1
hour and clarified cell lysate was flash frozen and stored at
-80.degree. C. prior to purification.
[0406] Clarified cell lysate was thawed and subsequently loaded
onto a 5 mL pre-packed HisTrap FF column equilibrated with 25 mM
HEPES, pH 7.5, 500 mM NaCl, 10% glycerol and 5 mM DTT. Proteins
were eluted with 500 mM imidazole at a flow rate of 1 mL/min.
Fractions containing the protein of interest were applied onto a
pre-packed 26/10 HiPrep Desalting Column equilibrated with 25 mM
HEPES, pH 7.5, 150 mM NaCl, 10% glycerol and 5 mM DTT. The
buffer-exchanged NS5B peak was then applied onto a 20 mL Poly-U
Sepharose column. Protein was eluted with an increasing salt
gradient and fractions collected. Protein purity was assessed on
Nu-PAGE pre-cast gels (Invitrogen, Carlsbad, Calif.). Purified NS5B
samples were concentrated using Centri-Prep concentrators
(Millipore, Billerica, Mass., USA) and protein concentrations were
determined by Bradford assay (Pierce, Rockford, Ill., USA).
1.b) Protein Sequence
[0407] PDB: 1nb4, Apo form
[0408] The protein sequence is as described in WO 2007/026024.
Calc. Mol. Properties: 64941.4 g/mol.
1.c) Inhibition Assay with NS5b 1bJ4
[0409] Measurement of HCV NS5B polymerization activity was
performed by evaluating the amount of radiolabeled GTP incorporated
by the enzyme in a newly synthesized RNA using heteropolymeric RNA
template/primer. The RdRp assay was carried out in 384-well plates
using 50 nM of purified NS5B enzyme, which was incubated with 300
nM 5'-biotinylated oligo(rG.sub.13)/poly(rC) or oligo
(rU15)/poly(rA) primer-template, 600 nM of GTP, and 0.1 .mu.Ci of
[.sup.3H]GTP or [.sup.3H]UTP in 25 mM Tris-HCl, pH 7.5, 5 mM
MgCl.sub.2, 25 mM KCl, 17 mM NaCl and 3 mM of DTT. The 30 .mu.L
reaction mixture was incubated at room temperature for 2 h before
stopping the reaction by adding 30 .mu.L of streptavidin coated
SPA-beads (GE Healthcare, Uppsala, Sweden) in 0.5 M EDTA. The 30
.mu.L reaction was terminated after 2 hours at 25.degree. C. upon
addition of 30 .mu.l streptavidin-coated SPA beads (GE Healthcare,
Uppsala, Sweden 5 mg/ml in 0.5 M EDTA). After incubation at
25.degree. C. for 30 min, the plate was counted using a Packard
TopCount microplate reader (30 sec/well, 1 min count delay) and
IC.sub.50 values were calculated (Table 1: IC.sub.50 1bJ4).
IC.sub.50 values represent the concentration of compound required
to decrease by 50% the amount of RNA produced which is measured by
the detection of incorporated radiolabeled GTP.
2. HCV NS5B con1b 2.a) Cloning, Expression and Purification of NS5B
con1b.
[0410] The coding sequence for NS5B (genotype 1b consensus strain
Con1) lacking 21 C-terminal residues was amplified from plasmid
pFKI.sub.389/ns3-3'_N (Genbank accession no. AJ242654) and
subcloned into the pET21b plasmid as described previously (Pauwels
et al, 2007, J Virol 81:6909-19). The NS5B.DELTA.C21 expression
construct was transformed into E. coli Rosetta 2 (DE3) (Novagen,
Madison, Wis.). One hundred milliliters of LB-medium supplemented
with carbenicillin (50 .mu.g/mL) and chloramphenicol (34 .mu.g/mL)
was inoculated with one colony, grown overnight, and transferred to
fresh LB-medium supplemented with 3% ethanol, carbenicillin and
chloramphenicol, at a ratio of 1:200. The remaining of the
procedure was as described previously (Pauwels et al, 2007, J Virol
81:6909-19), except that the column used for ion-exchange
chromatography was a 6 mL Resource S column (GE Healthcare), and
that protein concentrations were determined with the Nanodrop
(Nanodrop Technologies, Wilmington, Del., USA).
2.b) RNA-Dependent RNA Polymerase Assay.
[0411] Fifty-percent inhibitory concentrations (Table 1: IC.sub.50
con1b) were determined according to the method as described
previously (Pauwels et al, 2007, J Virol 81:6909-19) using a
primer-dependent transcription assay. Following a 10 minute
preincubation with the inhibitor, 20 nM of purified Con1b NS5B
enzyme was incubated for 10 min. with 150 nM 5'-biotinylated oligo
(rG.sub.13) primer, 15 nM poly (rC) template, 19 mM Tris-HCl, 5 mM
MgCl.sub.2, 17 mM NaCl, 21 mM KCl, and 2.5 mM DTT. 600 nM GTP and
0.13 .mu.Ci of [.sup.3H]GTP was then added to initiate the 40-.mu.l
reaction mixture, which was then incubated at room temperature for
2 h before the reaction was stopped by addition of 40-.mu.l
streptavidin-coated SPA beads.
[0412] The following Table 1 lists compounds according to any one
of the above examples. The activities of the compounds tested are
also depicted in Table 1.
TABLE-US-00001 TABLE 1 EC.sub.50 IC.sub.50 1bJ4 IC.sub.50 Con1b Nr.
Structure (.mu.M) (.mu.M) (.mu.M) 2 ##STR00185## = 0.09 = 0.41 1
##STR00186## = 0.07 = 0.24 = 0.038 11 ##STR00187## = 0.055 = 0.22 5
##STR00188## = 0.92 = 0.29 12 ##STR00189## = 0.29 = 0.93 10
##STR00190## = 0.06 = 0.28 13 ##STR00191## = 0.39 = 0.19 3
##STR00192## = 0.079 = 0.53 4 ##STR00193## = 3.83 = 2.42 6
##STR00194## = 0.550 8 ##STR00195## = 0.040 9 ##STR00196## = 0.800
15 ##STR00197## = 0.180 16 ##STR00198## = 0.078 = 0.040 17
##STR00199## = 0.170 18 ##STR00200## = 0.079 = 0.026 19
##STR00201## = 0.072 = 0.031 20 ##STR00202## = 0.081 = 0.038 21
##STR00203## = 0.280 22 ##STR00204## = 0.160 23 ##STR00205## =
0.470 24 ##STR00206## = 14.48 25 ##STR00207## = 0.550 26
##STR00208## = 0.130 27 ##STR00209## = 10.20 28 ##STR00210## =
0.330 38 ##STR00211## = 0.912
Enzyme Binding Affinity
[0413] The compounds of formula (I) were examined for their
enzymatic binding kinetics using a Surface Plasmon Resonance
(SPR)-based method, i.e. Biacore. A slow dissociation of the
inhibiting compound from its viral target (low k.sub.off, low
K.sub.d) is believed to potentially reduce the development of drug
resistance against anti-viral drugs (Dierynck et al. 2007. Journal
of Virology, vol. 81, No. 24, 13845-13851). All measurements were
performed on a Biacore T100 instrument (GE Healthcare). The
purified HIS.sub.6-tagged NS5B.DELTA.C21 polymerases were
immobilized using non-covalent capturing to an NTA sensor chip (GE
Healthcare) in immobilization buffer (20 mM MOPS pH 7.4, 500 mM
NaCl, 0.005% Tween-P20, 1 mM DTT, 50 .mu.M EDTA). Interaction
studies were all performed at 25.degree. C. Inhibitors were
serially diluted in running buffer (20 mM Tris-HCl pH 7.4, 150 mM
NaCl, 50 .mu.M EDTA, 1 mM DTT, 0.005% Tween-P20) containing 5%
dimethyl sulfoxide (DMSO). Single-cycle kinetics were used, in
which 5 increasing concentrations of compound were injected for a
period of 300 s each in 1 single cycle, and dissociation was
monitored for a period of 1200 s. The sensor surface was completely
regenerated in between the cycles. Data were analyzed using
simultaneous nonlinear regression analysis (global fitting) adapted
for single-cycle kinetics with Biacore T100 BiaEval evaluation
software 2.0 (GE Healthcare). The individual rate constants
k.sub.on and k.sub.off and a derived affinity constant,
K.sub.d=k.sub.off/k.sub.on, were determined by a kinetic evaluation
of the sensorgrams. The kinetic models accounted for bulk and
limited mass transport effects. Every analysis was performed at
least in two independent experiments. The dissociation rate of a
kinetic interaction can be translated into a compound residence
time (dissociative half-life t.sub.1/2=ln(2)/k.sub.off)
representative for the interaction time between the polymerase and
its inhibitor.
[0414] The observed association rate constants (k.sub.on),
dissociation rate constants (k.sub.off), derived affinity constant
(KO and dissociative half-life (t.sub.1/2) measured for compounds
of formula (I) or subgroups thereof on NS5B wild-type enzyme
(genotype 1b, Con1b) are given in Table 2.
[0415] Table 3 lists binding affinity data for compound nr. 1 on
different forms of HCV NS5B polymerase. The different forms studied
(NS5B Target) comprise different clinical isolates of different
genotypes of the wild type enzyme, and, different mutant NS5B
polymerases. The mutant enzymes were obtained by site directed
mutagenesis of the 1bJ4 or Con1b NS5B enzyme. Mutations P495L,
V494A and L392I are located in the binding pocket of the compounds
of the invention to NS5B polymerase.
[0416] It was observed that the strong binding of compounds of
formula (I) or subgroups thereof is consistent within one genotype,
that the compounds of formula (I) or subgroups thereof show
affinity for NS5B polymerase of the different genotypes, as well as
for NS5B polymerases with mutation in the indole binding pocket,
and that binding of the compounds of formula (I) or subgroups
thereof is not affected by mutations to other sites in the
enzyme.
TABLE-US-00002 TABLE 2 Nr. k.sub.on (1/Ms) k.sub.off (1/s) K.sub.d
(M) t.sub.1/2 (min) 16 2.2E+04 3.6E-05 1.6E-09 321.5 18 2.0E+04
4.8E-05 2.4E-09 241.0 1 2.0E+04 9.0E-05 4.4E-09 128.4 28 7.3E+03
6.6E-05 9.0E-09 175.5 25 2.9E+04 3.1E-04 1.1E-08 37.8 17 8.7E+03
1.6E-04 1.8E-08 72.0 27 9.5E+03 3.8E-03 4.0E-07 3.1 24 4.8E+03
3.7E-03 7.6E-07 3.1 38 5.3E+03 4.1E-05 7.7E-09 283.8
TABLE-US-00003 TABLE 3 NS5B target k.sub.on (1/Ms) k.sub.off (1/s)
K.sub.d (M) t.sub.1/2 (min) 1a isolate 1 7.4E+04 4.8E-04 6.5E-09
24.2 1a isolate 2 3.9E+04 3.7E-04 9.4E-09 31.3 1a isolate 3 8.1E+04
6.0E-04 7.4E-09 19.2 1a isolate 4 7.4E+04 7.7E-04 1.1E-08 14.9 1a
isolate 5 1.1E+05 3.1E-04 2.8E-09 37.2 1b isolate 1 2.6E+04 1.0E-04
4.0E-09 110.2 1b isolate 2 2.9E+04 6.7E-05 2.3E-09 172.1 1b isolate
3 3.7E+04 1.2E-04 3.3E-09 96.4 1b isolate 4 3.5E+04 1.7E-04 4.9E-09
67.5 2b isolate 1 1.8E+04 1.4E-02 8.2E-07 0.8 2b isolate 2 4.3E+04
1.2E-02 2.7E-07 1.0 2b isolate 3 4.4E+03 1.7E-02 3.8E-06 0.7 3a
isolate 1 9.5E+04 3.7E-04 3.9E-09 31.1 3a isolate 2 2.5E+04 4.7E-04
1.9E-08 24.6 3a isolate 3 6.0E+04 3.6E-04 6.1E-09 31.7 4a isolate 1
2.0E+05 4.3E-04 2.1E-09 26.7 4a isolate 2 2.8E+05 3.8E-04 1.4E-09
30.1 4a isolate 3 1.8E+05 6.0E-04 3.4E-09 19.1 5a isolate 4 4.3E+04
9.7E-04 2.2E-08 12.0 6a isolate 5 5.0E+04 1.6E-03 3.2E-08 7.3 1bJ4
2.0E+04 1.0E-04 5.2E-09 110.2 Con1b 2.0E+04 9.0E-05 4.4E-09 128.4
P495L (1bJ4) 5.9E+03 2.2E-02 3.8E-06 0.5 L392I (Con1b) 1.8E+04
9.7E-04 5.5E-08 11.9 P495L (Con1b) 3.4E+03 2.2E-02 6.4E-06 0.5
V494A (Con1b) 5.5E+04 8.4E-04 1.5E-08 13.8 M414T (1bJ4) 2.6E+04
1.5E-04 5.9E-09 75.4 M423T (1bJ4) 2.5E+04 1.5E-04 6.3E-09 75.0
S282T (1bJ4) 3.4E+04 1.3E-04 3.9E-09 88.9 C316Y (Con1b) 3.5E+04
7.6E-05 2.2E-09 151.7
* * * * *